In Assassin's Creed: Brotherhood, Ezio Auditore leads the Assassins in Rome.
Rome is the principal setting of Brotherhood and is controlled by the Borgia family at the start of the game. Ezio works to weaken Borgia power by eliminating key agents, freeing districts, recruiting new Assassins, and rebuilding the Roman Brotherhood. The city becomes a large connected environment rather than a brief stop between separate regions.
The central antagonist is Cesare Borgia, while his father, Rodrigo Borgia, is the Pope and a major figure in the wider conflict. The game uses Renaissance Rome's monuments and political institutions as backdrops for its fictional story, including the Colosseum, the Vatican area, and the Pantheon.
Florence and Venice are strongly associated with Ezio because they are major locations in Assassin's Creed II. Constantinople becomes the main city of Assassin's Creed: Revelations. Brotherhood's defining city, however, is Rome, and its liberation structure is one of the game's signature features.