The Sanctuary was accessed primarily through an entrance hidden behind a bookshelf in Mario Auditore's study in the Villa.
The Sanctuary was characterized notably by statues of seven legendary Assassins, who "guarded the freedom of humanity when it was most threatened."
Six of these statues each had corresponding seals at their base, which had to be collected and returned in order to unlock the gate in front of the seventh statue of Altaïr Ibn-La'Ahad. This seventh statue also held the armor set fashioned by Altaïr using the knowledge he gained from an Apple of Eden.
From left to right, the statues represented:
Siege of Monteriggioni
During the Fall of Monteriggioni, the Sanctuary was used as a temporary haven for survivors fleeing the attack, in which a passageway behind the statue of Altaïr served as an escape route for Ezio, his family, and several hundred of the townspeople.
Desmond Miles and Lucy Stillman traced back the escape route used by the townspeople after the siege in 1500, in order to unlock the bookcase entrance, and allow Rebecca Crane and Shaun Hastings to gain access to the chamber.
The modern Assassins then used it as refuge, since its underground structure was ideal in protecting them from Abstergo's cell phone surveillance. However, as the Sanctuary had no electricity, Desmond was tasked with navigating around modern-day Monteriggioni and rerouting power to the safehouse.
- The circular grate in the courtyard at the back of the Auditore Family Villa was actually the top of the Sanctuary, which allowed light in. The Sanctuary could not be viewed from the outside, however.
- In Assassin's Creed: Brotherhood, the grate at the top of the Sanctuary appeared broken from the inside, even though when viewed from the outside, it appeared undamaged.
- In Assassin's Creed II, all of the statues could be examined to gain information about the Assassin, with the exception being Altaïr's statue. In Brotherhood, only the statue of Altaïr could be interacted with, resulting in a short cutscene that involved Desmond using a stereotypical Italian accent, and Rebecca scolding his "racism." This process could have been repeated until Sequence 6, though without Rebecca's reply.
- Although all of the Assassin statues, other than Altaïr's, had the small skull mechanisms that opened the entrances of Assassin Tombs, they could not be interacted with.
- In Brotherhood, the skull mechanisms have been removed from the statues during the siege of Monteriggioni, but reappear in modern Monteriggioni.
- Desmond could interact with his teammates or certain items in the Sanctuary during each memory sequence, triggering a variety of conversations.