Stuart Gordon and Dennis Paoli originally intended to be faithful to H.P. Lovecraft's story, but the film ultimately has little in common with the story, which was intended to be a parody of "Frankenstein".
David Gale was made to shave his head and wear a toupee, as this was found to be in keeping with Dr. Hill's character. In the DVD commentary, it was revealed that this was also necessary for budgetary reasons, as there was no money available to match Gale's hair on a prosthetic head prop.
According to "Re-animator: Ressurectus", the 70-minute featurette on the Limited edition two-disc "Re-animator" box set, glowstick liquid was used for the glowing green "Re-agent". It is the first time glowstick liquid/glowsticks have ever been used on film.
In the DVD commentary, Jeffrey Combs expressed regret over the "Who's going to believe a talking head? Get a job in a sideshow." quote, mentioning that the "talking head" part got such a laugh out of theater audiences that the "sideshow" part (his personal favorite) often went entirely unheard.
Actor David Gale's wife divorced from him shortly after this film's release. In the DVD's audio commentary, the rest of the cast suspects that the scene when his character, Dr. Hill, attempts to rape Megan was the cause of divorce.
The "brains" in the severed head were made up of steer meat by-products, ground beef and fake blood and when they shot the scene in the autopsy room with the severed head being thrown out the door and then smashing onto the hallway wall, the crew were all behind the cameras with garbage bags over their clothes because no one knew just how much the brains would splatter.
The film loosely adapts the first half of Lovecraft's original short story, including Herbert and Dan (who is unnamed in the story) meeting in medical school, Dean Halsey's death and reanimation, and the decapitation and reanimation of an authority figure to the doctors ("Dr. Hill" in the film and "Major Sir Eric Moreland Clapham-Lee" in the story). The next film, Bride of Re-Animator (1990), loosely adapts the second half of Lovecraft's story, including the two doctors reanimating corpses on a battlefield (WWI in the story and the Peruvian Civil War in the film), West's experiments with reanimating individual body parts, West going beyond just stealing cadavers and resorting to murder to get fresh corpses, an outbreak of West's former experiments from an insane asylum, the decapitated villain's head being delivered to West in a box, and finally West being dragged by his experiments into a series of cemetery catacombs through a wall in his basement. The third film of the series is named Beyond Re-Animator (2003) because it literally goes "beyond" Lovecraft's original story.
The padded cell that Dean Halsey was confined in after being reanimated was hastily constructed and was prone to collapse. During early takes, actor Robert Sampson would dive into the walls while attempting to act insane and accidentally knock the walls over. Ultimately, Sampson was forced to not interact with the set and act out his scenes in the middle of the room, or lightly leaning against a wall. Evidence of the set's flimsiness can be seen when Halsey is fighting with two nurses. When he tosses one of them aside, the actor playing the nurse bumps into a wall which noticeably wobbles.
The bald, bearded doctor at the foot of Megan's bed who gets shoved away as Dan tries to revive her is underground cartoonist Kim Deitch ("The Boulevard of Broken Dreams"), son of legendary Jazz-era cartoonist Gene Deitch ("The Cat").
There was originally a subplot that revealed that Dr. Hill had the ability to control minds. It was cut from the film for timing reasons but evidence of it can still be seen in the story. He is seen performing this skill on both Megan and Herbert and is the reason he is able to control all of the zombies in the film's climax.