Assassin's Creed takes place in historical recreations of Venice

Similar to the first game, Assassin's Creed takes place in historical recreations of Venice, Florence, Forlì, San Gimignano, and the Tuscan countryside, and incorporates events during that period as part of the story. Missions are divided into main story missions, themselves divided into memory sequences reflected points along Ezio's life Cosplay Australia
, and side missions which can be accomplished at any time; this approach to mission structure remains consistent in the other games in the series. The Monteriggioni villa provides several functions which can be expanded on by paying money for upgrades of surrounding buildings, or by purchasing artwork, weapons, and costumes for the villa; in turn, the villa will generate wealth for the player at a rate influenced by the upgrades and acquisition of these items. Additional quests involve locating secret Assassin seals, and finding hidden marks left by "Subject 16", a former Animus user, that hint at the nature of Minerva's society.

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 Dead or Alive Costume
, who, through the use of the Animus, take on the genetic memories of Renaissance Templars in various game modes.

Desmond comes to consciousness in a virtual area within the Animus, the Animus Island, where he meets the digital personality of Subject 16. Subject 16 explains that from the trauma of stabbing Lucy, Desmond must complete the memories of both Altaïr and Ezio to be able to separate his mind from theirs and to allow him to come out of his coma; however, doing so will destabilize the island as the Animus recovers that memory segment and at one point Subject 16 sacrifices himself to allow Desmond to continue. Desmond rejoins Ezio's story many years after Brotherhood, where Ezio is curious to the Assassin's origins and has traveled to Masyaf to locate their original headquarters. He finds Altaïr's library, though it is locked by five keys which the Templars are also seeking, believing there to be great power within it. Ezio travels to the Ottoman-era Constantinople where the keys are said to have been hidden by explorer Niccolò Polo, and finds the city embroiled by the feuding brothers Selim and Ahmet vying for the Sultanate, the Templars secretly behind the battle. While searching for the keys, Ezio meets and falls in love with Sofia Sartor. Eventually Devil King Sanguko Basara II Costume
, Ahmet is revealed as the agent for the Templars, and is killed by Selim, who thanks Ezio for his help but banishes him from the city for his own good.