- Mrs. Carl O'Connor (voiced by Sally Struthers), the kindergarten teacher
- Mr. Carl O'Connor (voiced by Kevin Clash, Fred Stoller, and Tom Kenny), the kindergarten teacher's husband
- Bus Driver Bob (voiced by Fred Willard), the loyal bus driver
- Mr. Richard Warner (voiced by Tom Bosley), the principal at Lakeshore Elementary School
- Farmer Thomas Warner (voiced by Jorge Diaz), Richard Warner's brother and the owner of Warner Farms
- Daphne-Betsy's mother (voiced by Bess Armstrong), a homemaker who takes care of her daughter Betsy and her baby son Kevin. She is a stay-at-home mother and does not seem to have any outside job.
- Darnell-Betsy's father (voiced by Richard Steven Horvitz), an airplane pilot who speaks to Betsy in "airplane-talk." He appears to be the family's sole breadwinner.
- Betsy (voiced by Daveigh Chase), the title character of the show was a Game called "Learn with Betsy"
- Scott (voiced by Richard Steven Horvitz), a smart boy with glasses who is very interested in science was a Game called "Make Statues with Scott"
- Billy (voiced by Nancy Cartwright), who enjoys playing in the dirt and is Betsy's best friend was a Game called "Colors with Billy"
- Molly (voiced by Vicki Lewis), an elitist girl was a Game called "Listen to Sounds with Molly"
- Sarah (voiced by Cree Summer), a girl who excels at nearly all sports was a Game called "Exercise with Sarah"
- Kenji (voiced by Nancy Cartwright), an intelligent boy who enjoys dancing was a Game called "Learn parts of the body with Kenji"
- Maria (voiced by Cree Summer), a quiet and artistic girl was a Game called "Play Pretend with Maria"
- Kevin (voiced by Nancy Cartwright), Betsy's baby brother
- Newton (voiced by Kath Soucie), who shares similar interests to Scott and Kenji was a Game called "Watch the Video with Newton"
- Gracie (voiced by Kathryn Mullen), Betsy's family's dog
- Kitty (voiced by Pam Arciero), Betsy's family's cat
- Sidney (voiced by Anthony Asbury), the pet salamander that belongs to Mrs. O'Connor's kindergarten class
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