The narrator of this fictional autobiography is Cal Stephanides, an American of Greek descent with a hereditary 5-alpha-reductase deficiency that gives her the prepubertal anatomy (and thus the social upbringing) of a girl, but at puberty begins her transformation into ambiguity, then maleness, and then, gradually, masculinity.

The novel is a kind of biography, not just of Cal, but also of the mutant gene that causes her/his condition. It is transmitted from a small village in Smyrna, through his grandparents, who were also brother and sister and who married on the ship to America, apparently leaving behind family as well as national identity. Their Greekness and the gene come with them, and the consequences of their incest haunts Cal's grandmother, Desdemona, until the very end of the novel.

The family settles in Detroit, and a third biographical strand is the story of the Greek immigrant community in 20th century America, from Ford's assembly lines to bootlegging during the prohibition, through Detroit race riots and then to affluent suburbia.

Cal's family settles in the suburb of Middlesex, and the focus narrows to the individual. Calliope is raised as a girl, but in adolescence, Callie learns about hermaphroditism, narrowly escapes sex-assignment surgery, becomes a performer in a seventies sex show in San Francisco, and finally returns home to Middlesex, Grosse Point, Michigan, as a male. The story is framed by Cal's much later adult life as a man in Berlin, and his successful romance with a woman he meets there.