- Fredric March
- Samuel Langhorne Clemens, aka Mark Twain
- Alexis Smith
- Olivia Langdon Clemens
- Donald Crisp
- J.B. Pond
- Alan Hale
- Steve Gillis
- C. Aubrey Smith
- Oxford Chancellor
- John Carradine
- Bret Harte
- William Henry
- Charles Langdon
- Robert Barrat
- Horace E. Bixby - Riverboat Captain
- Walter Hampden
- Jervis Langdon
- Joyce Reynolds
- Clara Clemens
- Whitford Kane
- Joe Goodwin - Editor
- Percy Kilbride
- Billings - Enterprise Typesetter
- Nana Bryant
- Mrs. Langdon
- Ernie Adams
- Pickpocket
- Hooper Atchley
- Secretary
- Arthur Aylesworth
- Worried Buffalo Merchant
- Lynn Baggett
- Susie Clemens
- Leah Baird
- Elderly Woman
- Frank Baker
- Audience Member
- Brandon Beach
- Ceremony Spectator
- Willie Best
- George
- Edward Biby
- Dinner Guest
- Henry Blair
- Boy
- Oliver Blake
- Miner
- Monte Blue
- Captain of 'Queen of Dixie'
- Rudy Bowman
- Townsman
- Jackie Brown
- Samuel Clemens - age 12
- Bob Burns
- Passenger
- Viola Callahan
- Elderly Woman
- Burr Caruth
- Oliver Wendell Holmes
- Wheaton Chambers
- Joker at Train Station
- Davison Clark
- Henry Wadsworth Longfellow
- James Conaty
- Man in Audience
- Chester Conklin
- Frog-Jumping Contest Judge
- Carol Coombs
- Young Clara Clemens
- Lloyd Corrigan
- Town Citizen
- Joseph Crehan
- Riverboat Captain / Ulysses S. Grant (uncredited)
- Oliver Cross
- Audience Member
- Frank Dae
- Houghton
- Frank Darien
- Toy Store Proprietor
- Earle S. Dewey
- Southerner
- Dudley Dickerson
- Comet-Watcher
- Hazel Dohlman
- Audience Member
- Dennis Donnelly
- Baby
- Roland Drew
- Editor
- Bill Edwards
- Miner
- Sarah Edwards
- Townswoman
- Dick Elliott
- Townsman who challenges Mr. Lake / Laughing man in speech audience (uncredited)
- Jim Farley
- Prospector
- Franklyn Farnum
- Man in Audience
- Ross Ford
- Hardy
- Willie Fung
- Chinese Man
- Rosina Galli
- Italian Nurse
- Jack Gardner
- Townsman
- Jack Gargan
- Townsman
- Russell Gleason
- Orion Clemens
- William Gould
- Ship's Bouncer
- Mildred Gover
- Black Woman
- Jessica Grayson
- Housekeeper
- William Haade
- Steam Room Chief
- Frank Hagney
- Crew Chief
- Creighton Hale
- Man with Mule
- Thurston Hall
- Politician
- Sam Harris
- Dinner Guest
- George Haywood
- Englishman
- Robert Herrick
- English Boy
- Harry Hilliard
- John Greenleaf Whittier
- Gene Holland
- Huckleberry Finn
- Harry Holman
- Drunken Guest
- Stuart Holmes
- Man at Reception
- Robert Homans
- Policeman
- Olin Howland
- Riverboat Southerner
- George Humbert
- Townsman
- Brandon Hurst
- Ralph Waldo Emerson
- Charles Irwin
- British Reporter
- Kay Johnson
- Jane Clemens
- Payne B. Johnson
- Boy in Frog-Jumping Scene
- Dickie Jones
- Samuel Clemens - age 15
- Lew Kelly
- Pilot House Guest
- Fred Kelsey
- River-Rafter
- Bill Kennedy
- Townsman
- Victor Kilian
- Higgins
- Richard Kipling
- Englshman
- Al Kunde
- Townsman
- Bobby Larson
- Boy
- Peter Lawford
- Young Oxford Celebrant
- Billy Lechner
- English Boy
- George Lessey
- Henry H. Rogers
- Carl M. Leviness
- Man in Audience
- Wilbur Mack
- Man in Audience
- Charles Marsh
- Prospector
- Thomas Martin
- Oxford Professor
- Eric Mayne
- Audience Member
- Frank Mayo
- Pilot House Guest
- Charles McAvoy
- Prospector
- Merrill McCormick
- Townsman
- Sam McDaniel
- Comet-Watcher
- Sammy McKim
- Cub Pilot
- Robert Milasch
- Townsman
- John 'Skins' Miller
- Cross-Eyed Man
- Michael Miller
- Tom Sawyer
- Gloria Moore
- Young Susie Clemens
- Edmund Mortimer
- British Observer
- Jack Mower
- Pilot House Guest
- Richard Neill
- Audience Member
- Paul Newlan
- Boss Deck Hand
- Frank O'Connor
- Carriage Driver
- Pat O'Malley
- Captain's Aide
- Bud Osborne
- Lee Griggs
- Paul Panzer
- Pilot House Guest
- Charles Peck
- English Boy
- Frank Pharr
- Stagecoach Passenger
- Francis Pierlot
- Paige
- Lillian Randolph
- Clemens' Servant
- George Reed
- Comet-Watcher
- Frank Reicher
- Doctor in Elmira
- Betty Roadman
- Young Lady
- Christian Rub
- Jones
- Loretta Russell
- Audience Member
- Paul Russell
- Audience Member
- Charlene Salerno
- Young Jean Clemens
- Cliff Saum
- Prospector
- Francis Sayles
- Englishman
- Paul Scardon
- Rudyard Kipling
- George Sherwood
- Prospector
- Bhogwan Singh
- Audience Member
- Emmett Smith
- Repeater
- Walter Soderling
- Telegrapher
- Frederick Spencer
- Jim
- Count Stefenelli
- Ship Passenger
- Libby Taylor
- Black Woman
- Joyce Tucker
- Jean
- Harry Tyler
- Assistant Editor
- Dorothy Vaughan
- Katie Leary
- Sailor Vincent
- Shopkeeper
- Charles Waldron
- Dr. Quintard
- Eddy Waller
- Southerner
- Lee 'Lasses' White
- Yokel
- Leo White
- Barber
- Ernest Whitman
- Stoker
- Frank Wilcox
- Judge John Marshall Clemens
- Lottie Williams
- Lady in Audience
- Norman Willis
- Ben Lake
- Joan Winfield
- Young Lady
- Douglas Wood
- William Dean Howells
- Harry Woods
- Bixby's Depth-Caller
- Harry Worth
- Gambler
