The novel starts with a narration by its protagonist, Cal (his masculine identity), also known as Calliope (feminine): He recounts how 5-alpha-reductase deficiency, a recessive condition, causes him to be born with female characteristics. The book continues with accounts of his family's history, starting with his paternal grandparents in their home village and ending with his father's funeral. These accounts cover the conception of Cal, his teenage years, and the discovery of his intersex condition. Throughout the book, Cal weaves his opinion of the events in hindsight and of his life after his father's funeral. Eugenides sets Middlesex in the 20th century and interjects historical elements, such as the Balkan Wars, the Nation of Islam, the Watergate scandal, and the 1967 Detroit riot in the story.
The accounts of Cal's family history start from 1922. His grandfather, Eleutherios "Lefty" Stephanides, lives in Bithynios, a village in Asia Minor. Eugenides places the village high on the slope of Mount Olympos, above the city of Bursa, and describes incestuous marriages between cousins as a quietly accepted custom among the villagers. Lefty makes a living selling silkworm cocoons harvested by his sister, Desdemona. The siblings are orphans; their parents are victims of the ongoing Greco-Turkish War. As the war progresses, Lefty and Desdemona develop a romantic relationship. Fleeing the chaos brought by the war, they board a ship amid the Great Fire of Smyrna and set sail for the United States. Their histories unknown to the other passengers, they marry each other on board the vessel.
After arriving in New York, they locate their cousin, Sourmelina "Lina" Zizmo, in Detroit, Michigan, and stay with her. Lina is a closeted lesbian and the only person there to know of the siblings' incestuous relationship. Starting a new life, Lefty takes on a job at Ford Motor Company, but is later retrenched. He unknowingly joins Lina's husband, Jimmy, in bootlegging. Desdemona gives birth to a son, Milton, and later a daughter, Zoe. Lina gives birth to a daughter, Theodora or "Tessie". The relationship between Lefty and Desdemona declines after she learns that there is an increased chance of genetic disease for children born from incest. In 1924, after Milton's birth, Lefty opens a bar and gambling room, calling it the Zebra Room.
Milton and Tessie marry in 1946. They have two children, Chapter Eleven[note 3] and Calliope ("Callie"). Prior to Callie's birth, Desdemona predicts the child to be a boy, although the parents prepare for a girl. Chapter Eleven is a biologically "normal" boy; however, Callie is intersex. Her family members are unaware of her situation for many years, so they raise Callie as a girl. The family gets caught up in the 1967 Detroit riot, after President Johnson authorizes the use of federal troops. The family home is raided during this period, to the shock of the parents. After this harrowing experience, the family moves to a house on Middlesex Boulevard, Grosse Pointe.
When she is 14 years old, Callie falls in love with her female best friend, whom Callie refers to as the "Obscure Object".[note 4] In separate encounters, Callie has her first sexual experiences with a woman, the Obscure Object, and with a man, the Obscure Object's brother. After Callie is injured by a tractor, a doctor discovers that she is intersex. She is taken to a clinic in New York and undergoes a series of tests and examinations. After learning about the syndrome and facing the prospect of sex reassignment surgery, Callie runs away and assumes a male identity as Cal. He hitchhikes cross-country and reaches San Francisco, where he joins a burlesque show as Hermaphroditus.
Cal is arrested by the police during a raid on his workplace. He is released into Chapter Eleven's custody and learns of their father's death. The siblings return to their family home on Middlesex. In a private moment, Desdemona recognizes Cal's condition, associating it with stories from her old village about children born of incest. She confesses to Cal that her husband, Lefty, is also her brother. As Milton's funeral takes place at the church, Cal stands in the doorway of his family home, assuming the male-only role in Greek traditions to keep his father's spirit from re-entering the family home. Several years later, Cal becomes a diplomat stationed in Berlin. He meets Julie Kikuchi, a Japanese-American woman, and tentatively starts a relationship with her.