Desmond and his allies retreat to the ruins of the Monteriggioni villa, and Desmond reenters the Animus to continue Ezio's memories, specifically to identify the location of the Apple. After facing against Rodrigo Borgia, Ezio returned to Monteriggioni, but the villa is soon attacked by forces under the command of Cesare Borgia, Rodrigo's son. Mario is killed and the Apple is stolen. Ezio vows revenge by helping to free the people of Rome from the Borgia family. As Ezio works covertly to turn the city against the Borgias, he gains followers that want to join his cause, and Ezio trains them in the way of the Assassins. In an attempt to assassinate Cesare and Rodrigo at the Castel Sant'Angelo, Ezio instead witnesses Cesare forcing his father to eat a poisoned apple that Rodrigo had prepared to kill his son. Ezio chases down Cesare and eventually captures him and recovers the Apple. Cesare is taken to prison in Viana, Spain but manages to escape with help of his allies. The Assassins lead the fight against Cesare and the remaining loyal Borgia troops, and eventually Ezio throws Cesare from the castle walls, killing him. With no perceived further threats, Ezio takes to hide the Apple under the Colosseum. In the present, Desmond is able to navigate through the underground chambers beneath the Colosseum to locate the Apple. As he picks it up, Desmond is witness to another hologram figure, calling herself Juno, and who controls Desmond to stab Lucy (because Lucy has defected to the Templars) before he falls into a coma.
Brotherhood shares many of the same features as the previous game though it takes place primarily in Rome. Similar to the villa, the player is able to spend money to buy and upgrade shops and other facilities throughout the city as to increase revenue they can collect from it; however, the player will be required to destroy Borgia towers that control various sections of the city before they can do so. The Brotherhood of Assassins is introduced, by which, after saving citizens from certain events, the player can invite these citizens as Assassins; they can then be dispatched to remote locations across Europe to gain experience and money, or can be called in to help the player directly in a mission. For the first time in the series, the game features online multiplayer. Players play as Abstergo employees, who, through the use of the Animus, take on the genetic memories of Renaissance Templars in various game modes.