The film begins in a quarrell between two men at a remote mansion. Humbert Humbert (James Mason) a 40-something British professor of French literature, arrives at the masion which is ramsacked from an apparently wild party the night before and finds the insane and debauched Clare Quilty (Peter Sellers), whom is not only suffering from some kind of severe dementia, but is also clearly drunk, as he babbles on incoherently and does not seem to remember Humbert from a time ago. Quilty goes mad when Humbert points a gun at him. After a mutually exhausting struggle for it, Quilty, now insane with fear, merely responds politely as Humbert repeatedly shoots him. He finally dies with a comical lack of interest, expressing his slight concern in an affected English accent. Humbert is left exhausted and disoriented.
The film then turns to events four years earlier and goes forward as Humbert travels to Ramsdale, New Hampshire, a small town where he will spend the summer before his professorship begins at Beardsley College, Ohio. He searches across the town for room to let, being tempted by widowed, sexually famished mother, Charlotte Haze (Shelley Winters) to stay at her house. He declines until seeing her beautiful 14-year-old daughter, Dolores Haze (Sue Lyon), affectionately called Lolita (hence the title). Lolita is a soda-pop drinking, gum-chewing, overtly flirtatious teenager, with whom Humbert falls hopelessly in love.
In order to become close to Lolita, Humbert accepts Charlotte's offer and becomes a lodger in the Haze household. Soon, however, Charlotte announces that she will be sending Lolita to an all-girl sleep-away camp for the summer. On the morning of departure, Humbert receives a love confession note from Charlotte, asking Humbert to leave at once. The note says that if Humbert is still in the house when Charlotte returns from driving Lolita to camp, then he must join Charlotte in marriage. Humbert willingly marries Charlotte days later. After the wedding and honeymoon, Charlotte discovers Humberts diary entries describing his passion for Lolita, and has an emotional outburst. She threatens to leave forever, taking Lolita far away from Humbert. While Humbert hurriedly fixes martinis in the kitchen to smooth over the situation, Charlotte runs outside, gets hit by a speeding car, and dies.
Humbert drives to Camp Climax to pick up Lolita, who doesn't yet know her mother is dead. That night at a hotel, a pushy, abrasive stranger (Quilty) insinuates himself upon Humbert and keeps steering the conversation to his "beautiful little daughter," who is asleep upstairs. Humbert escapes the man's advances, and Humbert and Lolita enter into a sexual relationship. The two commence an odyssey across the United States, traveling from hotel to motel. In public, they act as father and daughter. After several days, Humbert tells Lolita that her mother is not sick in a hospital, as he had previously told her, but dead. Grief-stricken, she stays with Humbert.
In the fall, Humbert reports to his position at Beardsley College in Ohio, and enrolls Lolita in high school there. Before long, people begin to wonder about the relationship between father and his over-protected daughter. Humbert worries about her involvement with the school play and with male classmates.
One night Humbert returns home to find Dr. Zempf, a pushy, abrasive stranger, sitting in his darkened living room. Zemph, speaking with a thick German accent, claims to be a psychologist from Lolita's school and wants to discuss her knowledge of "the facts of life." Humbert is frightened and decides to take Lolita on the road again.
During the long drive across country, Humbert soon realizes they are being followed by a mysterious car that never drops away but never quite catches up. When they get a flat tire, Humbert sees the black car stoping beside the road not far from them. Lolita does not seem to be concerned about Humbert's suspicions that the man in the black car is following them, but when he offers to get out to talk to the driver, Lolita suddenly becomes nervous and tells Humbert not to engage the unseen driver in any conversation. Before he can make a decision at what to do, the black car turns around and drives away.
When Lolita becomes sick, Humbert takes her to a hospital in a small Arizona town. However that night, Humbert receives a phone call in his motel room from an unknown man about Lolita. Worried, Humbert returns to the hospital to pick her up, and she is gone. The nurse at the front desk tells him she left earlier with another man claiming to be her uncle and Humbert, devastated, is left without a single clue as to her disappearance or whereabouts.
Some years later, Humbert receives a letter from 'Mrs. Richard T. Schiller', Lolita's married name. She writes that she is now married to a nearly-deaf Korean War veteran named Dick, and that she is pregnant and in desperate need of money. Humbert travels to their home, where Lolita waits. Humbert finds that she is now 17-going-on-18, a roundly pregnant woman wearing eyeglasses leading a pleasant, humdrum life. Humbert demands that she tell him who kidnapped her three years earlier. She tells him it was Clare Quilty, the man that was following them, who is a famous playwright and with whom her mother had a fling in Ramsdale days. She states Quilty is also the one who disguised himself as Dr. Zempf, as well as the pushy stranger who kept crossing their path. Lolita claims that she herself carried on an affair with him and left with him when he promised her glamor. However, he then demanded she join his depraved lifestyle, including acting in his "art" films.
Humbert begs Lolita to leave her husband and come away with him, but she declines. Humbert gives Lolita $13,000, explaining that it's her share of the money from the sale of her mother's house. Leaving Lolita forever, Humbert surprises Quilty at his mansion where he kills him for abusing Lolita, thus bringing the film full circle. A disclaimer in the final shot reveals that Humbert died in prison of a heart attack while awaiting trial for Quilty's murder.