Junie B. is a plucky almost-six-year-old girl beginning her first days of kindergarten. She holds a highly developed sense of fairness and expresses a strong desire for independence. Anxious about new experiences like riding the loud and smelly yellow school bus, she struggles when adults ignore or dismiss her questions.

Mrs. is Junie B.'s kindergarten teacher who prioritizes a quiet and orderly classroom. She frequently forgets to use Junie B.'s middle initial. Her focus rests on compliance and shushing students rather than addressing the underlying causes of their conflicts.

Mother is Junie B.'s primary caregiver who struggles to manage her daughter's anxieties about starting school. She often brushes off Junie B.'s fears with quick reassurances and expects her to act like a "big girl" without explanation. She eventually tries to find practical solutions when she finally understands the extent of her daughter's panic.

Jim is a grouchy-looking kindergarten boy in Junie B.'s class. He reacts with hostility when Junie B. touches his backpack on the bus and continues to mock her during the school day. He acts as an immediate source of frustration for his peers.

Lucille is a kindergarten student in Junie B.'s class who wears bright "Very Very Berry" nail polish. She frustrates Junie B. by ignoring her claims to a red chair and laughing at her when Jim insults her. She also feeds her classmates' fears by sharing rumors about the bus ride home.

Principal is the head of Junie B.'s elementary school. He appears as an intimidating authority figure who expects strict adherence to the rules. When Junie B. hides instead of boarding the bus, he scolds her and rolls his eyes at her literal responses.

The school custodian manages the building's maintenance and carries the keys to the locked doors. He terrifies Junie B. by chasing her and calling her "Missy" during a bathroom emergency. He ultimately helps her inside to avoid an accident.

Grace is a kindergarten student who is scheduled to take the school bus for the first time on the second day of school. Mother sets her up as Junie B.'s bus buddy to help alleviate both girls' anxieties about the upcoming ride.