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Over its five seasons, The Ren & Stimpy Show featured a varied cast of characters, some who appeared in as few as one episode, and some who were practically regulars.

Ren Höek is a scrawny "Asthma-Hound" Chihuahua with a fairly long, rat-like, pink tail (which constantly disappears due to the character's animation limits) and tan fur. Martin "Dr. Toon" Goodman of Animation World Magazine described Ren as "scrawny," "dyspeptic," and "violently psychotic." On some occasions Ren "lost his mind" in a "cumulative process," resulting in Ren becoming, in Goodman's words, a "screaming klaxon, neon-pink eyes dilating into twin novae inches above his jagged, monolithic teeth." Andy Meisler of The New York Times described Ren as "adventurous," "intelligent," and "emotionally brittle."

John Kricfalusi originally voiced Ren in a manner that he describes as "a bad imitation of Peter Lorre." Billy West said that he auditioned to play Ren; the creators of the series believed that having West voice Ren and Stimpy would give him too large of a workload. West would, however, take over Ren's voice after Nickelodeon fired Kricfalusi. Kricfalusi would return for the Spike TV episodes and unaired episodes of The Ren and Stimpy Show. In the pilot, Pierre Decelles provided Ren's signature diabolical laughter, while West performed Ren's maniacal laugh in the series.

Kricfalusi complained about Nickelodeon executives requesting for Ren to have "a softer side."

Bill Wray said that Ren was his favorite character to write for; Wray described Ren as "fun" because "you can make him mean." In 1993 he added that, "It drives me crazy when I tell people I work on the show and they always say, 'Make Ren meaner'."

Stimpson "Stimpy" J. Cat is a fat, red and white Cornish Rex cat (described by George Liquor as a "Cornish Rex Hound" to enter him in a dog show) with a large blue nose, purple eyelids, no tail, white gloves with fingernails on them, human-style buttocks, flat feet and a brain the size of a peanut (despite some intelligence, such as when cooking and inventing). Martin "Dr. Toon" Goodman of Animation World Magazine described Stimpy as "obese" and "brain-damaged". Andy Meisler of The New York Times described Stimpy as "barrel-chested" and "good-natured".

Stimpy's trademark facial expression is a blissfully ignorant smile with his tongue flopping out. Most of the time when he gets excited, he says his famous catchphrase, "Oh, joy!" or simply "Joy". He also has a strong attachment to his "first material possession" which is a litter box (given to him in the pilot episode) and even though Ren destroys it (by bashing it on his head in "Fire Dogs" or dumping it in acid in "Ren's Bitter Half"), Stimpy always reacts strongly to it by crying, panicking or fainting, though it always appears again in some later episode. Stimpy is named after an art school classmate of Kricfalusi, whose nickname was "Stimpy Cadogen" ("Killer Cadogen" was Stimpy's pseudonym in several episodes, and in a few others he is referred to as Stimpleton Cadogen). Billy West, who voiced Stimpy, said that he based Stimpy's voice on a "sped up" Larry Fine of The Three Stooges. West described Stimpy as one of his favorite characters. Eric Bauza voiced Stimpy in the Ren & Stimpy "Adult Party Cartoon" because West believed that the series had no humor and that voicing Stimpy in it would damage his career, as West later said in an interview.

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