The overall story within Assassin's Creed is set in September 2012, where barkeeper Desmond Miles (the character controlled by the player) has been kidnapped by an unknown person prior to the start of the game, and is brought to Abstergo Industries, where researchers are working on the Animus project. The Animus is able to pull out memories from ancestors of the user, allowing the user to replay those memories as if he were there himself. Desmond was "chosen" for the project due to his relationship to his ancestor Altaïr, a member of the Assassins during the Third Crusade of the Holy Land; the researchers express interest in understanding more of how the Assassins worked. Though initially Desmond's sub-conscious rejects the memories of Altaïr, he eventually learns to accept them and is able to proceed to follow Altaïr's actions through the Animus.

The memories of Altaïr that Desmond experiences start in AD 1191: Richard the Lionheart has just recaptured the port city of Acre from occupying Muslims. With a base of operations established, the Crusaders prepare to march south. Their true target is Jerusalem – which they intend to recapture for Christianity. However, Saladin, leader of the Islamic army, currently rules the city. Stinging from his army’s defeat at Acre, he will not allow Richard to humiliate him again. The Muslim forces are massing in the ruins of Arsuf, intending to ambush the Crusaders and prevent them from reaching Jerusalem. These war maneuvers have left the rest of the Holy Land wide open. While Richard and Saladin battle one another, the men left to govern in their stead have begun taking advantage of their newfound positions of power. Exploitation, manipulation, and provocation rule the day.

The first memory Desmond experiences is that of Altaïr failing to assassinate the Grand Master of The Templar Knights, Robert de Sable, and recover the order's treasure. Due to this, Altaïr is demoted to Novice (the lowest rank in the Assassin Order). But Al Mualim, leader of the Assassins, offers him an opportunity to redeem himself. Altaïr must venture out into the Holy Land and assassinate nine men said to be exacerbating and exploiting the hostilities created by the Third Crusade. In doing so, he will stabilize the region, allowing Mualim to usher in an age of peace.


Cryptic messages on the wall of Desmond's bedroom at the end of the gameAlong the way, however, Altaïr discovers that his targets are bound by more than just a shared interest in personal gain, but are in fact Templars themselves, with the goal to unite all mankind under a common cause. Desmond further learns from emails when left overnight between sessions that Abstergo Industries is run by modern-day Templars, who are seeking the locations of several artifacts that they can use to continue to Templars' goals. Desmond learns that he was kidnapped by the company so that they may learn, through Altaïr's memories, where the location of an artifact may be buried in the Holy Land. He also finds that what remains of the Assassin's Guild in modern day had tried and failed to recover him before that information could be learned. Altaïr's memories finally reveal that the assignment he undertook was a ploy by the Templars to sow discord between the Assassins and the Crusaders and Muslims. In doing so, Saladin and Richard The Lionheart would instead work together and fight the Assassins, and in the process, bringing peace. Altaïr finally catches up with Robert de Sable, his last assigned target, and defeats him. de Sable reveals in his dying words that Altaïr's master is also a member of the Templars, and he has now the sole power of the artifact, the "Piece of Eden" that is able to alter what men can see and can be used for mass hypnosis. Altaïr travels to face his Master, who wields the "Piece of Eden" ,attempting to alter Altaïr's sight, but eventually falls to his blade. As Altaïr recovers the "Piece of Eden", the device activates one more time to reveal multiple locations across the globe in an holographic manner.

With Altaïr's memory complete, Desmond wakes up out of the Animus to learn that Abstergo Industries is already sending out recovery teams to those locations hoping to find additional artifacts. Desmond, no longer being of use to Abstergo Industries, was to be silenced; however, he was saved by Lucy. In the conclusion of the game, Desmond, having become "synchronized" with Altaïr, is able to use the eagle vision, and sees messages scrawled across the floors and walls that only he can see that refer to the end of the world described by several religions, among other writings; such writings include references to the biblical passage Revelation 22:13 ("I am the Alpha and the Omega, the first and the last, the beginning and the end."), a Lorenz Attractor, the Eye of Providence, and other writings in foreign languages. Additionally, there is the Mayan date of 13.0.0.0 — December 21, 2012 — which is only 3 months away within the game's timeline.