After the events of Assassin's Creed 2, Ezio Auditore flees to Monteriggioni and sets up a hideout in Villa Auditore, glad that his personal vendetta is finally over. However, the following day, the Villa is besieged by Cesare Borgia who steals the Apple of Eden and forces Ezio out of his home and into Rome, where the Templars are headquartered. In Rome, Ezio must revive the falling order of Assassins into a force that can challenge the Templar might - and have his revenge against Cesare Borgia.

In this direct sequel to Assassin's Creed 2, players once again play as Ezio as he continues his crusade against the Templars and restore the Assassins - forming what would become the Brotherhood.