Desmond's story continues where it left off, in 2012. 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.

Ezio's story continues in 1499, where he exits the Vault, confused by what he saw inside. He escapes Rome with his uncle Mario Auditore and arrives at Monteriggioni. Once home, Ezio is comforted by the prospect that his personal vendetta is over and that his days as an Assassin are done; however, Niccolò Machiavelli challenges Ezio's decision to leave Rodrigo Borgia, now Pope Alexander VI, alive. The following morning, Monteriggioni is besieged by Cesare Borgia, son of Rodrigo. Mario is killed at the hands of Cesare himself, and the Apple lost to Cesare and the Templars. Ezio escapes with his family and travels to Rome, the center of the Templar Order in Italy, once again seeking vengeance against the Borgias. There, he discovers that the Assassins are failing in the fight against corruption. Determined to redeem the Guild, Ezio convinces Machiavelli that he has what it takes to lead while assembling a Brotherhood powerful enough to destroy the Templars and his new arch nemesis, Cesare Borgia.

During the next four years, Ezio executes tasks meant to cripple the Borgias' hold in the capital, sabotaging Cesare's resources and assassinating key people close to and/or working with him in the process, and slowly restoring Rome to its former glory. Ezio is then made head of the Order and promoted to the rank of Il Mentore (Italian for "The Mentor"). During this time, Ezio's sister Claudia is made an assassin.

Cesare, learning of Ezio's actions, confronts his suspicious father and desperately asks for more money and the Apple itself. Rodrigo refuses and attempts to poison his son, realizing that Cesare's lust for power can't be held at bay. Cesare, however, turns the tables on his father and kills him instead. Ezio witnesses this and, after learning of the Apple's location, retrieves it from within St. Peter's Basilica. Ezio uses it to overwhelm Cesare's forces and withdraw the backing of his supporters; Cesare is finally arrested by Pope Julius II's Papal Army, after Ezio and the assassins confront him and kill his remaining followers.

Ezio uses the Apple once again to check on Cesare, who breaks out of prison and has lain siege on a small city in Navarre, Spain. He encounters Cesare on the battlements of a crumbling castle and fights him. Cesare claims that he cannot be killed by the hand of a mortal man, thus inciting Ezio to leave him "in the hands of fate" by dropping him off the battlements to his death. Ezio then takes the Apple and hides it in another temple built underneath Santa Maria in Aracoeli.

Using the coordinates taken from Ezio's memories, Desmond, Lucy, Shaun and Rebecca travel to the temple, intending to use the Apple to locate the remaining temples to keep the other Pieces of Eden out of the Templars' hands. As Desmond makes his way into the temple, he is confronted with holographic apparitions of a being called Juno, who appears to be of the same race as Minerva; however, she is neither audible nor visible to Lucy, Shaun, or Rebecca. Most of her comments center on humanity's lack of knowledge. She states that humanity is "innocent and ignorant"; that people were not built to be wise, having been given only five of the six senses: sight, smell, taste, touch, and hearing, but lacking knowledge. Her careful cadence then drops as she suddenly becomes angry, yelling "We should have left you as you were!"

As Desmond approaches the Apple and touches it, time freezes around him, though he can still move and speak. Juno cryptically says Desmond is descended from her race and their enemy; she also says there is a woman who would accompany him through "the gate," but must not be allowed to. She takes control of Desmond's body and forces him to stab Lucy in the abdomen. Both fall to the ground, with Lucy dead and Desmond entering a coma. As the credits roll, two men are heard discussing whether to put Desmond back in the Animus.
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