Stuart is a boy who looks exactly like a mouse, standing only slightly taller than a dime. He possesses a confident, adventurous personality and often dresses in tailored suits, complete with a tiny hat and cane. Despite his small stature, he refuses to be underestimated and actively seeks out large-scale adventures. He is resourceful and deeply loyal to his friends.

Margalo is a small brown bird with a streak of yellow on her breast. She comes from the wild, singing songs of fields and pastures. She is gentle and loyal, stepping in to help Stuart when he faces danger. Her presence brings a sense of the natural world into the city apartment.

Snowbell is a white household cat who prowls the neighborhood at night and sleeps indoors by day. He actively resents Stuart's status as a respected family member, feeling that a mouse should be prey rather than a brother. He masks his predatory intentions from the human parents while tormenting Stuart in private.

Frederick is Stuart's human father. He is a practical man who thoughtfully adapts the family home to suit his tiny son, crafting items like miniature ice skates and a tiny wooden mallet. He harbors a quiet yearning for exploration and takes pride in his son's physical freedom to explore small spaces.

Eleanor is Stuart's human mother. She is highly nurturing and goes to great lengths to ensure Stuart never feels alienated, even altering nursery rhymes that mention mice. Her maternal anxiety is a constant presence, as she frequently worries about Stuart falling down drains or getting trapped in the house.

George is Stuart's energetic, full-sized older brother. He eagerly accepts Stuart's unusual size and frequently devises elaborate mechanical schemes to help his brother move around the house. He has a short attention span and rarely finishes building his contraptions, leaving Stuart to find his own solutions.

Doctor Carey is a dentist whose true passion lies in sailing miniature boats and building small, motorized cars. He recognizes Stuart's capability immediately and treats him as an equal rather than a curiosity. He serves as a vital ally for Stuart's excursions in the city.

Harriet is a young woman who lives in Ames' Crossing and happens to be exactly Stuart's height. She is well-dressed, polite, and completely unbothered by minor inconveniences. She represents a rare opportunity for Stuart to connect with someone of his own scale.

Katharine is a bright, thoughtful schoolgirl in the town where Stuart briefly teaches. She treasures a small pillow stuffed with pine needles, which she keeps as a physical token of a perfect summer memory.