Narrated by Cal Stephanides, a 5-alpha-reductase Greek-American hermaphrodite identifying as a male, Middlesex tells the story of the Stephanides family's immigration to America and their assimilation. Book One begins in 1922 with Cal's grandparents, Desdemona and Lefty Stephanides, who are siblings living alone in a small village in Turkey, working as silk farmers. When the Turkish army invades Bursa and Smyrna, the siblings are forced to flee. They witness many atrocities as the cities burn down but, thanks to Lefty's courage and ingenuity, they manage to make it safely onto a ship that takes them to Greece. From Greece they board a ship to America. On that ship, they realize that they are in love with each other, and that no one knows they are actually siblings. They stage a fake courtship, and before the journey is over, they have been married in a Greek Orthodox marriage ceremony. Once in America, they contact their cousin Lina Zizmo, who lives in Detroit. They tell Lina their secret, which Lina agrees to keep because she also has something to hide (she is a lesbian).
Book Two tells of how Lefty and Desdemona adjust to living in America, as well as the story of Milton and Tessie, Cal's parents. Lina's husband, Jimmy Zizmo, gets Lefty a job at the Ford Motor Company. While there, Lefty graduates from their English school with flying colors. His success, however, is marred by investigation into his home life. The Ford authorities find out that Jimmy has a criminal record and they dismiss Lefty from work. Lefty now goes to work for Jimmy, helping him bootleg alcohol during Prohibition. One night, after the couples go see an erotically charged performance of The Minotaur, both couples have sex and conceive children. As the women cope with the stress of pregnancy, the men avoid the house. Jimmy begins to suspect (falsely) that Lefty and Lina are having an affair. One night, he lures Lefty onto the ice to threaten him. The plan backfires, and Jimmy crashes through the ice.
Desdemona gives birth to a son - Milton - and Lina gives birth to a daughter - Tessie - and the families hold Jimmy's funeral despite never finding a body. After learning about the genetic complications caused by incest, Desdemona fears giving birth to more children, so she begins to avoid Lefty. Lefty in turn starts a speakeasy in the basement of their house. Frustrated with being ignored for too long, Lefty grows angry with Desdemona, demanding that she get a job to support the family. She gets a job at a temple run by the new-found Nation of Islam, teaching the women how to raise silkworms. While there, she overhears their enigmatic leader, Minister Fard, preach on the dangers and treachery of white people. Emotionally vulnerable, Desdemona begins to take his words to heart and worries even more about her and Lefty's relationship. One day, the police arrest Minister Fard for inspiring another man to commit human sacrifice. He is forced to leave Detroit and as he walks past Desdemona, she realizes that Minister Fard is, in fact, the supposedly dead Jimmy Zizmo.
The narrative moves forward a few years to the middle of World War II, and Tessie and Milton are now young adults. Lefty has started a restaurant named the Zebra Room. Milton has begun to court Tessie, playing romantic songs for her on his clarinet. Fearing their "consanguinity," Desdemona tries to set the two cousins up with other people. She succeeds in getting Tessie to accept an offer of marriage from Michael Antoniou, a short, young man studying to be a priest. In protest, Milton enlists in the Navy. Once he gets there, he realizes what a mistake he's made and, afraid of going into combat, he desperately seeks a way out. Stuck at home watching movies while Michael is away at seminary, Tessie realizes that she actually loves Milton and writes to him, accepting his proposal. Desdemona agrees to it only because she thinks Milton is about to die. In a deus ex machina, Milton's application to Annapolis goes through, and right before he is to enter combat, he gets orders to report to the officer training academy.
Milton and Tessie get married and move around America for a little while. They have a son, whom they name Chapter Eleven. Father Mike marries Milton's sister Zoe. When Milton and Tessie finally move back to Detroit, Milton takes over Lefty's restaurant, making it very successful. Left without anything to do, Lefty starts gambling again, a habit from his days in Turkey. He eventually loses all his money, and Lefty and Desdemona are forced to move in with Tessie and Milton. Meanwhile, Milton and Tessie, wishing that they had a daughter, decide to try to conceive another child. Enter Calliope Stephanides.
Dr. Philobosian, an old family friend who fled Turkey with Desdemona and Lefty, delivers Calliope. He's too distracted by a beautiful nurse to realize that Calliope is not actually a girl, and so her family welcomes her home as the daughter they always wanted. As Calliope grows up, tensions in Detroit build between the races and riots eventually break out. Despite Milton's foolish efforts to physically protect his diner, someone burns it to the ground. The diner's fire insurance, however, saves the family and they end up getting a lot of money for it. They relocate to a wealthy suburb of Detroit named Grosse Pointe and Milton starts a roadside chain called Hercules Hot Dogs. There, seven-year-old Calliope makes a new friend, Clementine Stark, who practices kissing with her, awakening her sexuality. Lefty's health declines and a series of strokes results in his death and Desdemona's withdrawal from the outside world.
As the Stephanides grow more wealthy, Desdemona tries hard to die and Calliope wonders why she hasn't entered puberty yet. When Detroit passes a law to desegregate and "bus" its schools, Milton places Calliope in Baker & Inglis, an all-girls school. She feels uncomfortable changing clothes around her classmates and struggles with her body's development. Tessie takes her to a salon to remove the hair emerging on top of her lip. Meanwhile, Chapter Eleven comes home from college a hippie, high on LSD and toting his Marxist girlfriend behind him. He fights with the family, eventually dropping out of college to live in the backwoods of Michigan.
At school, Calliope is placed in the accelerated English class, where she meets a girl she calls "The Obscure Object." They are cast in a production of Antigone and they become friends. During the performance, another girl dies and, traumatized, the Obscure Object runs to Calliope for comfort. They grow closer over the summer and Calliope realizes that she loves the Object. The Object invites Calliope up to her summer house, with the Object's older brother Jerome and his friend Rex Reese. There, the four teenagers drink and smoke pot, and Jerome and Calliope have sex while the Object and Rex hook up next to them. Calliope is petrified that Jerome might have found something wrong with her, but he doesn't notice anything. The Object acts jealous towards Calliope the next morning, and the two girls begin a secret sexual relationship at night, which neither acknowledge during the day. One day, while Calliope is subtly stroking the Object's genitals, Jerome walks in on them and viciously makes fun of them for being lesbians. Calliope attacks him and then runs for her life. While she is running, she fails to see the tractor headed her way, and she crashes into it. As a car drives her off to the hospital, the Object kisses her goodbye, their first and last kiss.
At the hospital, the doctors realize that there is something wrong with Calliope's genitals and they recommend that her parents take her to a sexologist in New York named Dr. Luce. He is very famous, having redefined the criteria for determining gender. After examining Calliope for several weeks, he tells her parents that she is essentially a girl with male hormones kicking in at puberty, and that with hormone therapy and a little surgery, she can remain their little girl. Calliope, however, reads his written report and realizes that she is, biologically and genetically, a boy. Freaked out by this information, she runs away. Now going by Cal, (s)he hitchhikes across the United States, meeting kind people and creepy people along the way. Cal ends up in San Francisco, where he lives in a park with other runaways.
One night, while everyone else is at a Grateful Dead concert, other homeless men beat Cal up when they discover his gender secret. Penniless and bleeding, Cal calls the man who drove him into San Francisco, Bob Presto. Presto runs a strip club called Sixty-Niners, and he offers Cal a job portraying "the god Hermaphroditus" in the peep-show part. There, Cal meets other hermaphrodites who give him support and guidance. One night, the police raid the club and arrest Cal for being underage. At the police station, Cal calls his family and he learns that his father is dead. While searching for his missing daughter Calliope, Milton got a ransom call. He decided to meet the person without telling anyone, and after delivering the cash, he realized that it was actually Father Mike. Furious about the con, Milton chased Father Mike down and crashed the car on the Canadian border, dying on impact. Although saddened, Cal tells us that it is somehow for the best, since Milton missed many of the upcoming hardships. Cal returns home for Milton's funeral, but he stays behind to talk to the aging Desdemona. She doesn't recognize Cal at first, but eventually she realizes who Cal is and apologizes for her actions long ago. She tells Cal her secret, that she and Lefty were siblings. Cal promises not to tell people until after Desdemona dies.
In the present, Cal is living in Berlin, working for the Foreign Service. He meets an Asian woman named Julie Kikuchi, whom he takes on a couple of dates. Although he originally pulls away from her, afraid that she will reject him, he goes back and tells her the truth. She accepts him, and they tentatively and happily start their new life together.