The show tells the story of Anne Boonchuy, a self-centered 13-year-old who is magically transported to the fictitious world of Amphibia, a rural marshland full of frog-people. With the help of an excitable young frog named Sprig Plantar, Anne will transform into a hero and discover the first true friendship of her life.
- Brenda Song as Anne Boonchuy
- Justin Felbinger as Sprig Plantar
- Bill Farmer as Hopadiah (Hop Pop) Plantar
- Amanda Leighton as Polly Plantar
- Anna Akana as Sasha
- Tony Hale as Apothecary Gary and Lloyd
- Chris Sullivan as Gunther and Teddy
- Stephen Root as Mayor Toadstool
- Jack McBrayer as Toadie
- Kristen Johnston as Braddock
- Matt Jones as Percy
- Tara Lipinski and Johnny Weir as talent judges
- Diedrich Bader as Frog Jordan
- Kevin McDonald as Albus Duckweed
- James Patrick Stuart as One-Eyed Wally
- Troy Baker as Captain Grime
- Laila Berzins as Sadie Croaker
- Brian Maillard as Leopold Loggle
- Kevin Michael Richardson as Mr. Flour
- Jill Bartlett as Maddie Flour
- Dee Bradley Baker as Bessie
- Kaitlyn Robrock as Felicia Sundew
- Katie Crown as Ivy Sundew
- Mona Marshall as Sylvia Sundew
- Paul Eiding as Monroe
- John DiMaggio as Stumpy
- This is the first time Brenda Song has starred in a Disney Channel production since the ending of The Suite Life of Zack & Cody sequel, The Suite Life on Deck in 2011.
- Amphibia is one of the few non-action two-dimensional animated cartoons to have a human character animated with five fingers. The anthropomorphic amphibians having four fingers makes sense since real-life amphibians have four digits on their hands.
- The episode title cards depict a wood carving in darkness lit up with the title superimposed over it. This is an actual wood carving that is photographed and reused every episode.