The show was created by Jim Jinkins (the creator of Doug), and executive produced by David Campbell in close conjunction with Harvard University's Cognitive Skills Group, "Project Zero". The Group's job was to monitor each and make sure the material had a positive educational message. In that respect, PB&J Otter was one of the few shows of its kind on the air at that time.
It also aired on Playhouse Disney Channel Asia up until July 1, 2009. The show will air on Disney Junior channel in September 2012, along with the former Playhouse Disney shows.
- Peanut Otter (is voiced by Adam Rose) The brother of Jelly and Butter. About seven years old and eldest of the Otter children, Peanut is smart and can be a little funny at times, but always loyal. He hates dancing, especially "The Noodle Dance" but he still does anyway.
- Baby Butter Otter (is voiced by Gina Marie Tortorici) The baby of the family. She doesn't talk much, but says an odd word now and then.
- Jelly Otter (is voiced by Jenell Brook Slack) The middle of the Otter children, she often comes up with ideas that are creative but sometimes aren't feasible.
- Opal Otter (is voiced by Gwen Shepherd) - She is the wife of Ernest Otter and PB&J's mother. Opal is caring, but can be strict when necessary. She has purple hair in an afro and wears thick glasses and pink slippers.
- Ernest Otter (is voiced by Chris Phillips) - is PB&J's father and the husband of Opal Otter. He owns his own hardware store on Lake Hoohaw.
- Anna ('Aunt Nanner') Otter – Opal's sister. Has an unspecified job in show business, and travels the world. She knows many famous people and seems quite well off (but not nearly as rich as the Snooties), but always has time for her family. Married to her husband, Redolfo.
- Pinch Raccoon (is voiced by Cody Pennes) A friend of PB&J. She thinks of herself as beautiful and a ballerina, and spends most of her time dancing or making herself more pretty. She is Jelly's best friend.
- Pennes also voices Scootch Raccoon, Pinch's little brother. He is rambunctious and a bit of a trouble-maker to his friends and parents. He doesn't speak very much, generally only saying "Sorry, sorry, my fault" when he invariably knocks something over, but later in other episodes, he slowly becomes capable of forming new (but short) sentences. He makes a lot of mistakes by accident, but he still has a good heart and apologizes. Sometimes he feels left out due to his clumsiness, but everybody loves him, including Pinch, his sister. His age is unknown, but judging by his size, he may be 3 years old. He is Baby Butter's best friend.
- Munchy Beaver (is voiced by Chris Phillips)- A beaver who has quite the appetite for wood. He often tends to chew on things made of wood when he is nervous. He gets scared easily, and is rather jittery. He tends to have a low self-esteem. He is one of PB&J's friends.
- Cap'n Crane (is voiced by Chris Phillips) - A 'watchbird' which he calls himself. He keeps an eye on Lake Hoohaw and its population, in order to keep everyone safe, though he tends to overdo it from time to time, often being too safe for his own good, which tends to complicate things, though his heart is in the right place. His catchphrase is 'Watchbird Alert!'. He is Connie Crane's husband.
- Connie Crane (is voiced by Jackie Hoffman) Cap'n Crane's wife. She is usually seen sleeping and is often woken by her husband for unimportant alarms. She tends to think Cap'n Crane overdoes his job a bit and generally keeps an eye on him to keep him from making mistakes. She is very sarcastic most of the time.
- Flick Duck (is voiced by Eddie Korbich)- A duck who is sometimes clumsy and rude, but just like the others, he always apologizes for making mistakes. He is one of PB&J's friends. He also loves and can't resist babbleberry pie. His catchphrase is: "Cheese and quackers!".
- Korbich also voices Ootsie and Bootsie Snootie, son and daughter respectively of Edouard (also voiced by Korbich) and Georgina Snootie; rich, spoiled and generally bored, they nonetheless enjoy the company of the other children when they tire of playing with the newest toy or gadget their parents have bought them. Although they are mean to PB&J and their friends at times, PB&J and the others still help them when they have a problem. Ootsie and Bootsie can still be good friends and they also helped the kids many times.
- Mayor Jeff (is voiced by Bruce Bayley Johnson) - The mayor of Lake Hoohaw. He is a wise, old otter who loves to help out the kids. He also is known for his collection of various treasures. He is also known to babysit the Otter children from time to time.
- Walter and Wanda Raccoon (Walter voiced by Chris Phillips): - Pinch and Scootch's parents. Wanda is the post raccoon for Lake Hoohaw, while Walter works as the area trash collector, requiring him to sleep most of the day as he gets up very early.
- Shirley Duck (voiced by Corinne Orr): - Flick's mother. Very softhearted, she rarely so much as raises her voice to Flick, much less punishes him. Flick takes advantage of this whenever possible. Flick's father never appeared in the series and it is unknown if he has one.
- Betty-Lou Beaver: - Munchy's mother. She is proud of her son and tries to help him become more outspoken. Munchy's father has never been seen, but he has sent Munchy presents in the mail. It can be assumed he works away from home.
- Bubbles (voiced by Chris Phillips): The Otters' pet fish.
- Mr. Bigdog: - Edouard's boss.
- Measles: - Munchy's frog.
- Edouard Snootie: - Father of Ootsie and Bootsie.
- Georgina Snootie: - Mother of Ootsie and Bootsie.
- Billy Duck: - Flick's older cousin. Billy was once thought to be a bully by the Lake Hoohaw kids due to Flick's remembering something improperly, but he turned out to be kind. He may look scary, because he has a stern expression, nevertheless he still smiles and is kind to the ones around him. Like Flick, he likes to read Mallard Man comics.
- Ricky Raccoon: - Pinch's and Scootch's cousin. He is also kind, and he may be a little older than Pinch.
- Simon Fieldmouse: - A little field mouse boy who mistook Flick for the Ice Moose. Simon taught Flick to care about others.Flick dressed himself as the Ice Moose and took Simon to the stage where everybody, including Simon's parents, was there.
- Redolfo: Boyfriend (and now husband) of Annette Otter. Usually transports her to and from Lake Hoohaw.
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