Cal Stephanides is an intersex man living in Berlin, Germany. In an exercise of self-discovery, he decides to write his life story, starting with the history of the recessive gene that causes his intersex condition. The story begins with his grandparents in a tiny village on Mount Olympus in 1922. Lefty and Desdemona are siblings who have fallen in love with each other. After their parents’ deaths, they decide to immigrate to Detroit, where their cousin Sourmelina lives, and they head for the city of Smyrna to search for a boat to take them to mainland Greece. They escape the devastating burning of Smyrna by the Turkish army and find passage to America. On the ship, Lefty and Desdemona pretend to meet for the first time and then they wed. In Detroit, they move in with Sourmelina and her husband, Jimmy Zizmo. Zizmo takes Lefty into his alcohol smuggling business. After Desdemona and Sourmelina get pregnant on the same night, Zizmo worries that Sourmelina might be cheating on him with Lefty. He takes Lefty on a drive on the ice in order to interrogate him, but the car falls into the water. Lefty escapes.
In 1945, Lefty and Desdemona’s son, Milton, develops a crush on Zizmo and Sourmelina’s daughter, Tessie. Desdemona encourages Tessie to accept the proposal of a seminary student, Michael, causing Milton to join the Navy in a fit of rage. When Tessie realizes Milton will likely die in the U.S. invasion of Japan, she calls off her engagement to Michael. After excelling on a test, Milton receives a transfer to the officer’s academy and is spared fighting in the rest of the war. He and Tessie marry and have a son named Chapter Eleven. Five years later, both of them wish for a daughter, and Milton convinces Tessie to try to conceive a girl using pseudo-scientific methods for timing ovulation. The resulting baby, Calliope (Callie), will later become Cal. After retiring from the Navy, Milton takes over Lefty’s bar and turns it into a diner. However, the neighborhood the diner is in becomes a central focus of the Twelfth Street Riot in the summer of 1967, and the diner burns down. Because Lefty had taken out three insurance policies on the building, the family has enough money to move to the affluent suburb of Grosse Pointe. They move into an unusual house on a street named Middlesex.
Now seven years old, Callie is an arrestingly beautiful girl who desperately wants to make friends in her new neighborhood. The girl next door, Clementine Stark, invites her over and asks if they can practice kissing. Although Callie is too young to put words to it, her heart flutters. For high school, Tessie and Milton send Callie to an all-girls prep school to escape the governor’s orders to better integrate Detroit’s schools. Now nearly fourteen, Callie worries that she has not yet gotten her period or started to grow breasts. She begins to develop a moustache, and she grows the hair on her head long to hide her face. Soon, she develops a crush on a classmate that Cal, as narrator, refers to as “the Obscure Object.” When the girls are in a play together, Callie and the Object become friends, and the Object invites Callie to her family’s summer home. Eventually, she and the Object begin a physical relationship. When the Object’s brother, Jerome, realizes what has happened, he bullies his sister, and Callie attacks him. Callie flees and is hit by a tractor. At the hospital, the doctors realize that Callie is biologically male. Tessie and Milton don’t want to believe this is true and take Callie to a specialist in New York named Dr. Luce.
Dr. Luce is excited to meet Callie because he believes he can use her to prove his theory that gender is a social construct. Callie visits the library and looks up words she hears Dr. Luce use when he describes her to other doctors, which brings her to the words “hermaphrodite” and “monster.” Dr. Luce, deciding that Callie is a girl, recommends surgery to “fix” Callie’s genitals. When Dr. Luce isn’t looking, Callie peeks at her files. She learns that she’s biologically male and that surgery would likely cause her to lose sexual sensation. Horrified, Callie decides he’s a boy, renames himself Cal, and runs away to San Francisco. After mishaps on the road and sleeping in Golden Gate Park, Cal finds work at a peep show that displays people with ambiguous gender. Here, he meets Zora, another intersex person, who teaches him that he’s not alone. In Detroit, Cal’s parents are devastated and desperate to find their child. When the police raid the peep show, Cal calls home and learns that Milton has died in a car accident that occurred when he tried to catch a person who claimed to have kidnapped Callie. This person turns out to be Father Mike, the man Tessie left for Milton years ago. Cal returns home for the funeral but opts to talk with Desdemona instead of attending. Desdemona confesses that she committed incest and apologizes for the gene she and Lefty passed to Cal. Cal tells her he will live a good life. Years later, Cal starts a relationship with a woman named Julie Kikuchi in Berlin.