Duck awakes from a dream in which she is a duck watching a handsome prince dance ballet. Later, she walks early into class and discovers Mytho, a student in the advanced class, dancing alone. She has a nerve-racking conversation with him before Fakir, another student, walks in. Fakir is revealed to be domineering and condescending towards Mytho. In ballet class, we learn that Mytho is dating a girl named Rue. The teacher is a cat, yet Duck is the only one in the class to find this strange. Rue is revealed to be a talented girl in the advanced class, who most of the students look up to. After class, Duck goes to the boy’s dorm to apologize to Mytho for accidentally causing him to injure his leg. Before she can work up the courage to go in, Fakir stops her and turns her away. Later that night, Duck regrets not being able to apologize to Mytho, and ponders his lonely eyes. She wishes to help him, catching the attention of the mysterious Drosselmeyer, who says to himself: “As the water flows, time has begun to move. Now, let me listen to your story!” The next morning, Fakir goes to the library. As Duck resolves to apologize to Mytho, she spies canary chicks on the dorm roof beginning to fly. One of the fledglings hesitates, and is forced to jump when a raven attacks it. Mytho leaps to catch the bird, and falls as well. To save him, Duck transforms into Princess Tutu. The bird flies away, and Mytho asks who she is. Tutu/Duck remembers that she is a duck, and runs away. The story has begun.

The next day, Duck wakes up to realize that she is just a duck. Drosselmeyer appears to tell her that she is also the legendary Princess Tutu; her task is to return the pieces of heart to the Prince, Mytho. Longing to one day see Mytho smile, she decides to take up the challenge. Drosselmeyer gives back the pendant that changes her into a girl, as well as Princess Tutu. An Anteater-girl, who is bitter and jealous of Rue for her superior dancing skills, sets her eyes on Mytho. She and Mytho have a dance-off with Rue. Rue chooses Duck as her partner and they win. Duck discovers that the anteater is being influenced by a shard of Mytho's heart. She transforms into Princess Tutu and convinces the heart shard to return to him. Mytho regains a feeling, which worries Fakir.

Duck learns about the book The Prince and the Raven. She discovers that its author is Drosselmeyer, and that he died a long time ago. She bumps into Mytho, who is retrieving water for Rue, while he and Rue were on a picnic. Duck uses up the water to clean a cut Mytho got, and while on a search for more water, the two stumble upon a restaurant. Inside, a woman, Ebine, serves them cold food and Duck discovers that she has another piece of Mytho’s heart: the feeling of loneliness. She retrieves it and afterwards Rue (and Fakir, who had been looking for him) discuss Princess Tutu, and her fate. The audience learns that if Princess Tutu confesses her love to the Prince, she will turn into a speck of light.

Duck contemplates who she is as Princess Tutu. Mytho notes that someone is calling him, and empathizes with the feeling that that someone wants to meet someone, recognizing the feeling to be loneliness. Fakir tries to convince Mytho that he does not need a heart; meanwhile, Duck gets chastised for interrupting Rue’s practice rehearsal. Duck vows to return Mytho’s heart to him, while denying that she loves him. Later, Rue reiterates Fakir’s words to Mytho, telling him that he loves her, and that is all he needs to know. Fakir tries to find out why Mytho is regaining his feelings. Mytho follows the aforementioned call and disappears. Duck and Rue go looking for him. They run into Edel, who tells them a story and directs them in the right direction, which enables them to find Mytho. He has found the ghost Giselle, who has been calling to him, and is offered a rosemary flower, which will allow Giselle to take him with her to the land of the dead. Rue tries to save him by dancing with Giselle, but fails. When Rue falls, Duck hides and transforms into Princess Tutu. Tutu reveals herself to Rue and dances with Giselle. She successfully gains the feeling of sadness from Giselle and gives it to Mytho. Drosselmeyer notes that the feelings returned to the Prince, thus far, are unhappy ones, and asks Tutu, “is this how you will restore happiness to the Prince?”

Rue looks through The Prince and the Raven, and is confronted by Fakir. She hints that Princess Tutu may be real. Meanwhile, Duck learns about the Fire Festival. Duck has been charged with cleaning the room of the advanced class. She arrives at the classroom and finds Mytho, dressed in Festival garb, awaiting Rue to practice together. Since she is absent, he decides to dance with Duck instead, to her joy. Fakir sees the two and confronts Mytho, telling him to just listen to him. The two leave and Rue appears. The two talk for a few moments, and Duck goes out in search of Mytho. As punishment for not listening to him, Fakir locks Mytho in a closet. Outraged, Duck tries to open the locked door, and lets slip that she knows that Mytho is regaining the pieces of his heart. Fakir becomes suspicious and questions her. Duck discovers that both Rue and Fakir know about Mytho being the Prince from the story. Fakir aggressively tells Duck that Mytho does not need a heart. In the closet, Mytho hears a voice talking to him from a strange glowing panel in the floor. Edel appears once more, and points out an open window that Duck can go through. She does and she falls onto the glowing panel, which opens and causes her to tumble down into some catacombs. She goes in search of Mytho, and a voice begins telling her riddles. She gets them all wrong, but finds the room Mytho is in, regardless, and gets locked in the room. She transforms into Princess Tutu, and finally gets that the answer to the riddles, revealing the voice as a lamp with a piece of heart. Tutu dances while the lamp spotlights her. The lamp reveals that she once shone for people, but was cast aside. She now wants Mytho so that she can shine for him. Tutu talks the lamp into releasing Mytho, and promises to take the lamp with her. Mytho’s feeling of affection is returned to him. Rue, waiting for Mytho at the plaza even after the festival ends, finally turns to leave but finds Mytho in her path. They dance while Duck

A traveling ballet troupe comes into town and Duck's whole class gets time off to go and see the troupe practice. Duck sees that a heart shard has taken possession of the lead in the upcoming Sleeping Beauty show, but it leaves when the void in her heart is filled when the lead's husband encourages and supports her. Duck chases after the shard and asks it to return to Mytho, but it instead attacks her with a sword. Despite its obvious reluctance, Duck catches it and returns it to Mytho. The shard turns out to be the feeling of fear and uncertainty, and once it is returned Mytho becomes afraid of Princess Tutu. Duck is shocked and dismayed that her attempt to help Mytho has ended with him in pain.

Distraught over what she has done to Mytho, Duck thinks about giving up being Princess Tutu, but knows that if she does she can no longer be a girl. Drosselmeyer is worried that the story will be frozen again if Duck quits, so he stops time to encourage her. His pep talk, however, achieves the opposite of his intentions: Duck throws her pendant into the river, transforming back into a duck. Meanwhile, Mytho is confused at the new emotions he is feeling and wanders to the river. There, the heart shard of curiosity takes him deep underwater. Duck tries to save him but cannot in her duck form, so she retrieves her pendant and becomes Princess Tutu once more. Mytho tells her that he does not want her to go; that he wants her to restore his heart. Gladdened, Tutu is about to restore his feeling of curiosity when it is suddenly stolen by the Raven Princess, Kraehe.

Rue wakes up and cannot remember what happened the night before. She finds Mytho's heart shard in one of her drawers. It questions her and asks her who she really is, but she is unable to answer. Duck ponders whether or not to tell Mytho she is Princess Tutu. Mytho defies Fakir, who, realizing he can no longer control Mytho, retrieves the sword of the prince to break Mytho's heart into pieces again. Kraehe (an unwitting Rue) appears and tries to steal Mytho from Princess Tutu, and then Fakir appears and tries to break Mytho's heart. Princess Tutu is able to stop him, and she also returns the heart shard of curiosity. However, she despairs of Mytho finding out she is in reality just a duck.

Fakir, having failed to shatter Mytho's heart, wonders what he should do: should he give up, especially now since Mytho wants to restore his heart? Rue has her own demons to deal with, as she tries to convince herself that there is only one of her. But Rue does not want Mytho to change, so she decides to accept her role as Kraehe. Duck finds another piece of Mytho's heart, devotion, possessing a student artist. Princess Tutu removes the heart shard, but Kraehe appears and takes it from her. Mytho comes and Kraehe's resolve wavers when the one she loves stands against her, giving Princess Tutu enough time to restore the heart shard.

Kraehe retreats for the moment, and Fakir lunges at Princess Tutu with a shard of glass, missing but cutting the chain of her pendant. Princess Tutu turns into a duck, and by the time she realizes the pendant is missing, it is gone. She searches for it all night and finds that Fakir is keeping it. She spends the next day trying to get it back, finally succeeding at dusk. Meanwhile the audience learns that Fakir is an orphan, and his adoptive father a sword smith. Fakir tells his father that he has realized he is the reincarnation of the knight from the story the Prince and the Raven, but is afraid because it is the knight's fate to die, torn in half by the raven. Duck sees that Fakir's father is possessed by the heart shard of regret, and goes to recover it. Now free of regret, Fakir's father gives the knight's sword to Fakir and tells him to follow his heart. When returning to the school, Fakir sees Duck and notices the pendant she is wearing, and realizes that she is Princess Tutu.

Mytho, inspired by an overheard conversation between Duck and her two friends, decides to give a present to Princess Tutu to express his feelings and to find out how she feels. He goes out to town with Rue, where they find Edel selling gems. Edel gives Mytho a gem called Love, but warns him that although beautiful, it can be dangerous and delicate. Rue, aware of the gem's intended recipient, is jealous and scuffs it, then tells Mytho she will shine it and make it even more beautiful. Instead, she curses the gem with hatred and spells a raven's feather inside before giving it back to Mytho. Mytho gives it to Princess Tutu, who discovers the heart shard of love is inside. She is about to return it when Kraehe's hatred manifests and binds her. Kraehe appears and rips the half-returned heart shard out of Mytho's chest.

Kraehe and Mytho have vanished. Princess Tutu helps Fakir back to his dorm and binds his wounds, and when he wakes up they decide to search together for Mytho, though Fakir still refuses to team up with Duck. They search everywhere but find nothing, until they happen upon Edel, who shows them the way. In the passage, Duck is so clumsy that Fakir wonders if she and Tutu can even be the same person. She trips and he moves to save her, and they both ending up falling down a deep hole. Duck reveals to him that she is actually a duck and transforms to find a way to get out. Together, Duck and Fakir go to where the final stage has been set.

Duck and Fakir arrive at the stage where Kraehe is keeping Mytho, and Kraehe says she will give the shard of love back if Tutu confesses her feelings, knowing full well that if Tutu does she will turn into a speck of light and vanish. Tutu is prepared to sacrifice herself but Fakir stops her. He fights his way through the ravens in order to break the sword of the prince so Mytho's heart can never be shattered again. Tutu resolves to go on, and she decides to express her feelings through dance. Mytho is moved by Tutu's dance and he and the heart shard go to her, and his heart is repaired. The stage vanishes and they are lost in darkness, but a flame appears to show them the way out. The flame is Edel, who has set herself on fire to guide them back to safety. Edel makes a last request to Tutu and Mytho, that she might see them dance together. Princess Tutu and the Prince dance a duet, while Drosselmeyer muses on how they were able to overcome their tragic fate through much pain.