The game begins where Brotherhood left off. Present day protagonist Desmond Miles' mind has suffered a breakdown from the events of the previous games, and to save Desmond's mind, the Assassins put him into a specialized safe area of the Animus. Upon awakening inside the Animus, Desmond meets Subject 16, who occupied the Animus before him. 16 explains that Desmond's mind is fragmented, and that the only way for him to separate the pieces of his true self from those of his ancestors is to continue to play out their stories until there is nothing left for them to show Desmond. Once he reaches that point, the Animus will be able to separate the three individual's memories.
Desmond enters a memory portal, which places him in the perspective of Ezio Auditore. Ezio has traveled to Masayf to attempt to unlock the secrets Altair had previously discovered and to find the true purpose to his order. Upon arriving he finds the stronghold overrun by the Templars, who mark him for death. Ezio escapes to the bowels of the fortress where he discovers the entrance to Altair's library. He learns that five keys are required to unlock the door, that the Templars have the first, and that the rest lie in Constantinople. Upon recovering his first key, Ezio learns that each key is encoded with Altair's memories by having an Animus-like experience in which he relives key moments of Altair's life.
Upon arriving in Constantinople, Ezio is greeted by Yusuf Tazim, the leader of the local Assassin Order and also befriends a young student named Suleiman. Ezio learns that the Templars have already recovered one of the keys, and the rest were hidden in the city by Niccolo Polo. While searching for Niccolo's old trading post, Ezio comes across and befriends Sofia Sartor, a young Italian traveler and book collector, and he quickly becomes infatuated with her. Ezio has Sofia help him track down the locations of the remaining four keys, while keeping his true intentions secret from her. Meanwhile, Constantinople is in chaos due to conflicts between Prince Ahmet and his brother Selim, who are quarreling over who will inherit the Sultanate. Caught in the middle of the conflict, Suleiman reveals to Ezio that he is an Ottoman prince and that he suspects the Templars are behind Ahmet and Selim's feud. Ezio eventually uncovers evidence that Manuel Palaiologos, with Templar support, is attempting to raise an army to overthrow the Ottomans and reestablish the Byzantine Empire. Ezio kills Manuel and recovers the final key, only to discover that Ahmet is the true mastermind of the Templar plot to find Altair's library. Realizing that Ahmet knows about Sofia, Ezio races back to Constantinople.
With the five keys in his possession, Ezio manages to witness Altair's life after the events of the first game. After killing Al-Mualim, Altair takes possession of the Apple of Eden and assumes leadership of the Assassins. However, one of the Assassins, Abbas, does not support Altair. When Altair and his wife Maria Thorpe leave Masayf for ten years to repel the Mongol invasion, Abbas stages a coup, seizing control of the Assassins and executing Altair's youngest son. Altair returns to take revenge on Abbas, but Maria attempts to stop him. Abbas then takes the chance to kill Maria. Altair is forced to flee and go into exile for the next twenty years, in which time Abbas' paranoia and madness weakens the Assassins and undoes many of the gains they had made against the Templars. Altair finally returns to Masayf and manages to convince the Assassins to turn against Abbas. He then kills Abbas and takes back his rightful place as the leader of the Assassins. Years later, having seen the future through the Apple of Eden, an elderly Altair encodes his memories into the keys and entrusts them to Niccolo.
Meanwhile, inside the Animus, the entire environment begins breaking down as the Animus begins deleting unnecessary data. Subject 16 sacrifices himself to prevent Desmond from being deleted himself, giving him enough time to escape through the memory portal.
Back in Constantinople, Ezio discovers that Ahmet has killed Yusuf and kidnapped Sofia, demanding the keys for her life. Ezio agrees to the trade, but immediately gives chase to Ahmet after he ensures that Sofia is safe. He manages to recover the keys, but before he can do anything about Ahmet, Selim arrives with his armies and executes Ahmet himself. Because Ezio is a personal friend of Suleiman, Selim decides to spare Ezio's life but warns him to leave Constantinople and never return. Together, both Ezio and Sofia return to Masayf, where Ezio explains the true meaning of the Assassin's Creed and uses the keys to unlock Altair's library hidden underneath the castle. Upon entering, Ezio finds the library empty except for Altair's skeleton and a sixth key. Taking the key, Ezio discovers that the library does not hold Ezio's book, but the Apple of Eden (the one Ezio possesses is apparently a different one). Altair sealed himself inside the library in order to preserve its secret. Ezio decides to leave the Apple in the library, and begins talking to Desmond, not knowing exactly who he is but knowing that he is watching. He tells Desmond of his intention to finally retire from life as an Assassin, believing that he has served his purpose. He expresses his hope that Desmond will be able to find the answers to the questions that he and Altair had suffered so much to uncover.
Suddenly, Desmond is approached by Jupiter, another hologram similar to Juno and Minerva. He explains to Desmond that the First Civilization built numerous underground vaults that would independently study various methods to save the planet from destruction, with all of the data being transmitted to a central vault where he, Juno, and Minerva tested them. However, none of their methods were effective and they failed to stop the solar flare that destroyed the First Civilization. Jupiter continues telling Desmond that he has the power to save the planet from the second solar flare, and shows him the location of central vault. Desmond then finally wakes up from the Animus to see Rebecca, Shaun, and his father standing over him. They had managed to smuggle him out of Rome and used the coordinates from the Animus found at the end of the Da Vinci Disappearance to take him to the central vault. Desmond's arm then glows blue and upon seeing Ezio's Apple of Eden, tells the other Assassins that he knows what to do. Underground, the central vault activates.
