The premise behind Sister Princess is that an ordinary young man (the player) is made to live with twelve lovable little sisters, each with their own distinct quirks and personalities. Players have about a month to spend with the sisters with each day broken up into three segments: first is the free time period before noon, followed by the period after lunch and ending with another period of time before bed. During day time, the player can choose which of the girls he wishes to escort to school, if any. The period after lunch is similar to the one in the morning, with additional choices which allow the player to go to various places in town besides walking home with one of the girls. Every night before bed, the player will be allowed to check his e-mail and send replies. As the player continues to interact with the sisters, their reactions to their big brother will change accordingly until the ending.

There are two different endings for each sister: the normal ending and the "non-blood relation" ending. As the name would suggest, the special ending reveals that the brother-character and sister are not actually related by blood. In the case of some of the older sisters, this can lead to the protagonist and "sister" marrying one another.