A. C.

Desmond Miles, a bartender, is kidnapped by Abstergo Industries. There, Desmond is forced to interface with the Animus, a device that is able to replay the genetic memories of the user's ancestors. In Desmond's case, they seek information about his ancestor Altaïr Ibn-La'Ahad, an Assassin during the time of the Third Crusade. Within the Animus, Altaïr's memories reveal that he was attempting to stop Robert de Sablé from taking an artifact from a temple, but broke all three of the Assassin Brotherhood's tenets in the process. The Brotherhood leader, Al Mualim, demotes Altaïr to the rank of Novice, and assigns him the task of assassinating nine people, all of whom are Templars, to regain his former status.

Desmond Miles is still trapped by Abstergo Industries (the modern-day face of the Knights Templar) after being forced to use the Animus device to revisit genetic memories of the Assassin Altaïr Ibn-La'Ahad, and has discovered prophetic warnings from Subject 16 (a previous captive) describing the end of the world. He is soon rescued by Lucy Stillman, a mole for the Assassins within Abstergo, who takes him to meet two other Assassins, historian Shaun Hastings and computer expert Rebecca Crane. They request that Desmond use their version of the Animus, dubbed the Animus 2.0, to relive memories of another Assassin, Ezio Auditore da Firenze, to train Desmond in the ways of the Assassins through the "Bleeding Effect" of the Animus.

After escaping the Templars' attack at the end of Assassin's Creed II, Desmond Miles, Lucy Stillman, Rebecca Crane, and Shaun Hastings flee to Monteriggioni, establishing a new hideout in the ruins of the Villa Auditore. After restoring the electricity in the old tunnels under the villa, the player once again takes control of Ezio Auditore through Desmond's genetic memory, using the Animus 2.0 (which is the game's memory interface). Their mission is to find the Apple of Eden, an important and mysterious artifact that could prevent the impending disaster that is coming in that same year, believed to be perpetrated by the Templars.

Samples taken from Desmond Miles' body in the moments after his death have enabled Abstergo Industries to continue to explore his genetic memories using the Animus' newfound cloud computing abilities. The unnamed player character is hired by Abstergo Entertainment to sift through the memories of Edward Kenway, an eighteenth-century pirate, the father of Haytham Kenway and the grandfather of Ratonhnhaké:ton. Ostensibly, this is to gather material for an Animus-powered interactive feature film, but in reality, Abstergo—the Templars of the present time—are searching for a First Civilization structure known as the Observatory, and are using the memories of Edward Kenway to find it.

France's defeat caused the city of New Orleans to be taken over by the Spanish government. The citizens were upset over the transition from French to Spanish control and Spain's new occupation within the city. However, in 1765, the French governor Jean-Jacques Blaise d'Abbadie made a negotiation with Templar Rafael Joaquín de Ferrer to stay as governor of New Orleans. This plot was later uncovered by the Assassin Aveline de Grandpré, who infiltrated the governor's mansion and assassinated him.