In order of appearance:
- Huckleberry Finn is a boy about thirteen or fourteen years old. He has been brought up by his father, the town drunk, and has a hard time fitting into society. Tom Sawyer and his friends occasionally call him "Huck Finn".
- Widow Douglas is the kind lady who has taken Huck in after he helped save her from a violent home invasion. She tries her best to civilize Huck, believing it is her Christian duty.
- Miss Watson is the widow's sister, a tough old spinster who also lives with them. She is fairly hard on Huck, causing him to resent her a good deal. Samuel Clemens may have drawn inspiration for her from several people he knew in his life.
- Jim is Miss Watson's big, mild-mannered slave. Huck becomes very close to Jim when they reunite after Jim flees Miss Watson to seek refuge from slavery, and Huck and Jim become fellow travelers on the Mississippi River.
- Tom Sawyer is Huck's best friend and peer, the main character of other Twain novels and the leader of the town boys in adventures. He is "the best fighter and the smartest kid in town".
- "Pap" Finn, Huck's father, is the town drunk. He is often angry at Huck and resents him getting any kind of education. He also returns to Huck whenever he needs more money for alcohol.
- Judith Loftus plays a small part in the novel — being the kind and perceptive woman whom Huck talks to in order to find out about the search for Jim — but many critics believe her to be the best female character in the novel.
- The Grangerfords, an aristocratic Kentuckian family headed by the sexagenarian Colonel Saul Grangerford, take Huck in after he is separated from Jim on the Mississippi. Huck becomes close friends with the youngest male of the family, Buck Grangerford, who is Huck's age. By the time Huck meets them, the Grangerfords have been engaged in an age-old blood feud with another local family, the Shepherdsons.
- The duke and the king are two otherwise unnamed con artists whom Huck and Jim take aboard their raft just before the start of their Arkansas adventures. They are featured prominently throughout the novel, duping many local townspeople with their various cons. The middle-aged "duke" claims to be the long-lost Duke of Bridgewater (though he mistakenly says "Bilgewater" and is sometimes called this by the king), while the elderly "king" claims to be the long-lost Dauphin of France, and so is sometimes called "Capet" by the duke.
- Doctor Robinson is the only man who recognizes that the King and Duke are phonies when they try to pretend to be British. He warns the town but they ignore him. A different character of the same name in The Adventures of Tom Sawyer was the man who wanted a grave to be dug up, but Injun Joe subsequently murdered him and framed Muff Potter for that crime.
- Mary Jane, Joanna, and Susan Wilks are the three young nieces of their wealthy guardian, Peter Wilks, who has recently died. The duke and the king try to steal the inheritance left by Peter Wilks, by posing as Peter's estranged brothers from England.
- Aunt Sally and Uncle Silas Phelps, are the two people whose nephew Huck poses as, after he abandons the duke and king. She is a loving, but high-strung lady, and he a plodding old man, both farmer and preacher.
Many other characters play important but minimal roles in the many episodes that make up the novel. They include slaves owned by the various families they meet, supporting townspeople, rafts-men, a doctor and a steamboat captain.