Little Sister is first seen with her husband on Friar Tuck's scalp, cheering for Robin Hood and Maid Marian. She is then seen dancing on Friar Tuck's head as everyone present at a dance party makes fun of Prince John.
Next, she is shown in Friar Tuck's church, sweeping up the little Welcome mat outside the mouse hole, complaining over Prince John taxing the heart and soul out of the peasants. She then gets her (and Sexton's) last farthing (which they both have been saving) from under the mattress of their bed (which is a single shoe) and gives it to Friar Tuck for the poor. As the Sheriff of Nottingham enters, Sexton rudely asks what he wants, but Little Sister shushes him. She then scolds the Sheriff for entering to take the coin for Prince John by telling him to put it back, only for him to say that "His Majesty also blesses [her]". The two mice then watch Friar Tuck and the Sheriff fighting, only for the latter to arrest the former for "high treason to the crown" thanks to Trigger, causing Little Sister to cry into her apron as her husband is comforting her.
Finally, she and Sexton are seen at the end of the film, where the two celebrate newlyweds Robin Hood and Maid Marian by throwing rice after King Richard finally returns.