Will Smith as Oscar, an underachieving bluestreak cleaner wrasse who works in the Whale Wash of Southside Reef and has three longtime best friends named Angie, Nelson, and Sabrina who he has known for five years and he is Nelson's roommate. He wants to be rich and famous, but his schemes always fail and he ends up in-debt to his boss with five thousand clams.
Robert De Niro as Don Edward Lino, a great white shark and leader of a mob consisting of criminally-inclined great white sharks, who wants his two sons and only daughter to take over his business and run it together, but becomes enraged when Oscar inadvertently gets in the way following one of his children's death.
Renée Zellweger as Angie, an angelfish who is one of Oscar's best friends and receptionist of the Whale Wash as well as Sabrina's roommate and childhood best friend. Angie is secretly in love with Oscar and eventually becomes his girlfriend.
Neil Patrick Harris as Nelson, a tuna who is one of Oscar's best friends as well as his roommate and the leader of the Whale Wash dental squad. Nelson is secretly in love with Sabrina just like Angie secretly has a huge crush on Oscar, and eventually becomes her boyfriend.
Julia Louis-Dreyfus as Sabrina, a sea goldie who is one of Oscar's best friends and the assistant receptionist of the Whale Wash as well as Angie's roommate and has been best friends with Angie since childhood. Sabrina is also Nelson's love interest and eventually becomes his girlfriend.
Jack Black as Lenny Lino, Don Lino's middle child who is a vegetarian, the younger brother of Frankie, the older brother of Teresa, and becomes good friends with Oscar.
Scarlett Johansson as Teresa Lino, Don Lino's youngest child and only daughter, and the younger sister of Lenny and Frankie. Unlike her father and oldest brother, she accepts Lenny for who he is, and also becomes good friends with Oscar.
Angelina Jolie as Lola, a seductive lionfish who is a gold digger. Oscar was romantically interested in her until he eventually realized that she was shallow, vain, vengeful, and selfish. She only cares about money, calling herself superficial, and she is shown slamming Oscar into the windows.
Martin Scorsese as Sykes, the pufferfish owner of the Whale Wash and a loan shark to whom Oscar owes five thousand clams. He once worked with Don Lino, but was fired and called in his debts to pay off the mobster.
Ziggy Marley and Doug E. Doug as Ernie and Bernie, two Jamaican jellyfish and Sykes' enforcers, who enjoy torturing Oscar with their painful stingers when he is in trouble with their boss.
Michael Imperioli as Frankie Lino, Lenny and Teresa's older brother, and Don Lino's eldest and more vicious child, who is embarrassed of Lenny's vegetarian views unlike Teresa. Frankie is killed after an anchor accidentally hits him.
Vincent Pastore as Luca, Don Lino's octopus consigliere, with a tendency to state the obvious much to Lino's frustration.
Mindy Sterling as Yvette, Don Lino's barracuda caporegime, who constantly gets annoyed by Luca's foolishness just like Don Lino.
Peter Falk as Don Ira Feinberg, an elderly leopard shark and leader of a mob of criminally-inclined leopard sharks, who is a friend of Don Lino.
Emma Thompson as Don Judith Hammond, a thresher shark and leader of a mob of an all-female criminally-inclined thresher sharks, who is also a friend of Don Lino.
Katie Couric as Katie Current, the local reporter of Southside Reef.
David P. Smith as Crazy Joe, a deranged hermit crab who is Oscar's friend. He lives in a dumpster near the Whale Wash.
David Soren as Horace, a shrimp who fears being eaten by a shark and is an enemy to Don Lino.