Still in limited release 35 years after its premiere, it has the longest-running theatrical release in film history. The film is considered a cult classic. It gained notoriety as a midnight movie in 1977 when audiences began participating with the film in theatres across the United States. Rocky Horror is the first film from a major studio, such as 20th Century Fox, to be in the midnight movie market. Widely known by mainstream audiences, it has a large international following and is one of the most well known and financially successful midnight movies of all time. In 2005, the film was selected for preservation in the United States National Film Registry by the Library of Congress as being "culturally, historically, or aesthetically significant."

The story begins in the aftermath of a marriage ceremony attended by Brad Majors (Barry Bostwick) and Janet Weiss (Susan Sarandon), a young pair of lovers who are quickly inspired to become engaged and have their own wedding. A criminologist (Charles Gray) then makes his introduction as narrator, explaining the story according to a leather bound report titled, "The Denton Affair." After the ceremony, Brad and Janet, on their way to visit an old high school teacher, find themselves lost and with a flat tire on a cold and rainy, late November evening. Seeking a phone with which to call for help at a nearby castle, Brad and Janet encounter Riff Raff (Richard O'Brien), a hunch-backed butler, and his sister, Magenta (Patricia Quinn), a maid. These two tell Brad and Janet that they have arrived, coincidentally, on a very special night, revealing a group of strange and outlandish people holding an Annual Transylvanian Convention in the castle ballroom. Brad and Janet, still wet from the rain, watch as the "Transylvanians", servants, and a tap dancing groupie, Columbia (Nell Campbell), dance the "Time Warp", the film's signature song.

They are soon swept into the world of Dr. Frank-N-Furter (Tim Curry), the master of the castle and a bizarre, self-proclaimed "sweet transvestite from Transsexual, Transylvania". Frank claims to have discovered the "secret to life itself". In a scene inspired by the classic Frankenstein films, his creation, Rocky Horror (Peter Hinwood), an astoundingly muscular young man, is brought to life in the castle laboratory. The ensuing celebration is soon interrupted by Eddie (Meat Loaf), an ex-delivery boy and partial brain donor to Rocky, and Columbia's implied lover, who rides out of a deep freezer on a motorcycle. In a jealous rage, Frank lures him back into the deep freezer and kills him by hacking him with a pick-axe. He then departs with Rocky to a bridal suite connected to the laboratory.

Brad and Janet are shown to separate bedrooms, where each is visited and seduced in turn by Frank, posing as the opposite. Meanwhile, Riff Raff torments Rocky until he at last flees his room, allowing Riff Raff to make Frank believe that his creation has escaped. Janet, upset and emotional, wanders off to look for Brad, who she discovers is with Frank via a television monitor. She then discovers the mentally childlike but physically brawny Rocky, cowering in his birth tank, hiding from Riff Raff's bullying. Rocky, acting like a wounded animal, allows Janet to tend to his cuts and bruises and warms up to her. Moved by his vulnerability and willingness to be cared for by her and attracted to his physique, Janet seduces him and they proceed to have sex while she sings a song detailing her suppressed sexual frustration and the subsequent freedom granted by willingly giving herself over to pure lust, implying she is now losing her virginity. Meanwhile Magenta and Columbia watch voyeuristically from their bedroom monitor, mocking her and singing along in a jeering chorus.

After being told that Rocky has gone missing, Frank, Brad, and Riff Raff return to the lab. There, they discover Rocky naked in his tank with Janet, though this sexual revelation is interrupted when they learn that an intruder has entered the building. The German-accented Dr. Everett Scott (Jonathan Adams), Brad and Janet's old high school science teacher, has come looking for his coincidental nephew, Eddie, but Frank suspects Dr. Scott of working for the government, investigating UFO activity, and thus revealing he is an alien. Upon learning of Brad and Janet's prior acquaintance to Scott, Frank suspects them of conspiring with him the entire time.

With tension in the air, Frank serves Rocky and the guests meat for dinner, which, when Frank pulls off the tablecloth, is revealed to be part of Eddie's mutilated body. Janet runs screaming into Rocky's arms, and is slapped and chased through the halls of the castle by Frank, who can no longer suppress his jealous rage. Janet, Brad, Dr. Scott, Rocky, and Columbia all meet in Frank's lab, where Frank captures them with the Medusa Transducer, a device that transforms them all into stone statues (including Columbia, who rebels against Frank's authority). They are then all reanimated in drag-clothing that matches Frank's and with self-moving legs resembling Frank's, and forced to perform a live cabaret floor show under a sort of enchantment that makes them feel more open to Frank's hedonistic style and attitude.

The performance, concluding with all performers splashing around in bewitched joy in Frank's pool, is interrupted by Riff Raff and Magenta. These two, dressed in authentic Transylvanian garb, stage a mutiny armed with anti-matter laser guns and announce their plan to return to the planet of Transsexual, in the galaxy of Transylvania, without Frank as their leader. In the process they shoot and kill a screaming Columbia and an escaping Frank effortlessly, but Rocky seems resistant to the anti-matter beams as he rushes over to his dead creator, cradling Frank's head in dismay. Rocky proceeds to carry Frank's body while climbing up a set piece on stage (à la King Kong), being shot at by Riff Raff, though the structure topples over into the pool, where Rocky finally dies and his and Frank's bodies float lifelessly.

Riff Raff offers his condolences to Dr. Scott for his nephew's death, releases the humans, Brad, Janet, and Dr. Scott, and then departs with Magenta by lifting off in the Castle itself. Brad and Janet are left weak and corrupted from their adulterous encounter with the Transylvanians, having each sexually cheated on the other, their lives forever changed.

The film concludes with the Criminologist narrating the end of the case.

The original American productions of the stage musical were both produced by Lou Adler who is best known for his Cheech and Chong feature films. Adler brought the production to the US in 1974 and later became executive producer of the film version.