Lucy returns and breaks Desmond out of Abstergo. She brings him to a hideout and introduces Desmond to her Assassin allies Shaun and Rebecca. She also reveals that the Assassins are in possession of an updated version of the Animus. Aware of the Templars' intentions to gather the Pieces of Eden, Desmond volunteers to enter the Animus again, and explore the memories of Ezio Auditore da Firenze, a young nobleman from 1476 in Florence, Republic of Florence. He hopes to gain Ezio's abilities through the Bleeding Effect so he can aid the Assassins and destroy the Templars.



Ezio's father and brothers are executed by the hand of Uberto Alberti, a corrupt official in league with the Templars. Ezio kills Uberto in revenge, and after fleeing with his mother and sister to their villa in Monteriggioni, he learns of his Assassin heritage and begins training under his uncle Mario. Ezio also befriends local inventor Leonardo da Vinci, who helps Ezio with his work by decoding an encrypted codex that contains Altaar's memories, as well as plans for advanced assassin weaponry. As Ezio looks at them with Eagle Vision, the thirty codex pages come together to form a hidden map of the vaults across the world. Over the course of a decade Ezio assassinates the conspirators involved in his father's death, leaving Rodrigo Borgia, a nobleman and Templar who has retrieved the Apple of Eden and is the center of the conspiracy. Ezio and other Assassins reclaim the Apple of Eden, but Borgia flees. Ezio is formally inducted as an Assassin, and is told that he may be a prophet as described in the codex.



During the course of the game Desmond experiences a number of unusual occurrences. First he discovers strange glyphs, apparently left by Subject 16, a test subject before Desmond who died because the Abstergo staff left him in the Animus for too long. The glyphs are similar to the symbols he'd seen at Abstergo, which hide a secret history of the world. He later dreams of appearing as Altaar again, chasing after Maria, a Templar whom he'd previously spared, and romanced. Finally, several memories are skipped, throwing the timeline forward by more than a decade to the Vatican, where Borgia now resides as Pope Alexander VI. The "skipped" memories are available as downloadable content, and deal with the Battle of Forla and the Bonfire of the Vanities.



Borgia is now the Pope and has in his possession the Papal Staff, another Piece of Eden. He attempts to use it to enter a vault that the prophet as described in the codex must enter. Ezio intercepts Borgia, but spares his life, and enters the vault himself. He encounters the hologram of a woman, who claims that she'd once been known as Minerva, and who also claims to come from a civilization whose members were also seen in the same light by ancient peoples. Minerva tells of a great disaster that had befallen and destroyed her civilization, and warns that it will occur again soon. Talking directly to Desmond, she tells him "The rest is up to you," leaving Ezio extremely confused. Desmond is pulled out of the Animus as Abstergo has located their hideout. He, Lucy and the rest of the team manage to flee to safety, and soon prepare to return Desmond to the Animus to search for clues that may help them.