Celeste is a timid but highly resourceful mouse with tawny-colored fur and deep brown eyes. Living under the floorboards of the plantation's dining room, she spends her time weaving intricate baskets out of dried grasses. She is gentle and compassionate, often finding herself targeted by the rats who share her living space. Her desire for safety and beauty prompts her search for a secure, permanent home.

Joseph is a gentle, fifteen-year-old boy from Cincinnati who serves as Audubon's artistic assistant. He has chestnut hair, pale blue eyes, and struggles with intense homesickness and loneliness. Highly critical of his own botanical drawings, he doubts his artistic abilities but possesses a deep empathy for the animals he encounters.

Audubon is a tall, imposing, and highly ambitious naturalist staying as a guest at Oakley Plantation. He possesses a thick shank of auburn hair and an unyielding drive to paint lifelike portraits of every bird species in North America. To achieve his artistic goals, he hunts and poses birds using wire armatures, causing ideological friction with those who prefer to leave nature unharmed.

Trixie is a large gray rat with a raspy voice who lives under the floorboards of the plantation house. She is exceedingly greedy, cruel, and impatient. Instead of foraging for herself, she bullies smaller creatures and demands that they gather human food scraps for her, showing no regard for their safety.

Cornelius is a curious and thoughtful wood thrush with a yellow bill and creamy white breast feathers spotted with dark brown. Originally living in the nearby woods, he finds himself confined to a cage in Joseph's room. He possesses a beautiful, melancholy singing voice that inspires the artists working nearby.

Lafayette is a majestic osprey with golden yellow eyes, dark brown wing feathers, and a friendly, jovial demeanor. He spends his time flying along the river and speaks with a cheerful, colloquial dialect. Observant and altruistic, he is quick to offer help to smaller creatures traversing his territory.

Illianna is a gray rat who resides under the plantation floorboards alongside Trixie. Like her companion, she is aggressive and demanding, frequently stealing from Celeste and forcing the smaller mouse to act as a lookout during dangerous foraging trips into the dining room.

Dash is Mr. Audubon's large brown hunting dog. Described as being as big as a dining-room chair, he accompanies his owner on excursions to track and retrieve birds.

Mr. Pirrie is the owner of Oakley Plantation. He acts as a host to Audubon and his assistant, providing them room and board, but shows immediate hostility toward the mice that inhabit his grand home.

Mrs. Pirrie is the wife of the plantation owner. She maintains the grand dining room and is easily startled by the presence of rodents near her elegant dining table.

Eliza is the young daughter of the plantation owners. As part of her family's arrangement with their guests, she receives instruction in drawing, painting, and dancing from the demanding Audubon.

Ellis is a kindly groundhog who lives on the plantation grounds. He acts as a guardian to a young Celeste after her initial home is destroyed, bringing her to the safety of the tunnels beneath the large house.

Violet is a friendly wren who seeks shelter in the plantation house. She acts as a companionable presence, eager to share berries and listen to stories about the surrounding world.