A home on a hillside in Nagasaki, at the turn of the twentieth century. Goro, a Japanese matchmaker, shows American naval lieutenant B.F. Pinkerton the house that Pinkerton has decided to lease. The house comes with a bride, and it is time for the arranged wedding. Sharpless, the U.S. Consul in Nagasaki and Pinkerton’s guest, warns Pinkerton not to take advantage of the innocent, trusting Cio-Cio-San. But Pinkerton ignores him and toasts the day when he will wed a real American wife. Cio-Cio-San arrives with her relatives. She tells Pinkerton she has renounced her religion for his. The marriage is finalized, and relatives and friends are celebrating when Cio-Cio-San’s uncle, the Bonze, bursts in. Furious at Cio-Cio-San’s conversion, he ostracizes her from the family, and everyone abandons her. Left alone with her new husband, Cio-Cio-San is comforted by the possibility of happiness in her new life, and she and Pinkerton express their love.
Three years have passed since Pinkerton’s departure from Nagasaki. Cio-Cio-San's servant Suzuki warns they have almost no money and are near destitution, but Cio-Cio-San is confident that Pinkerton will return for her. Sharpless enters, bearing a letter from Pinkerton. He doesn’t get a chance to read it; Cio-Cio-San constantly interrupts him, and then Goro arrives with Prince Yamadori, a new suitor for Cio-Cio-San. According to Goro, under Japanese law Pinkerton’s absence is grounds for divorce. Cio-Cio-San replies that since she is American, Japanese law doesn’t apply to her.
After Goro and Yamadori depart, Sharpless bluntly asks Cio-Cio-San what she will do if Pinkerton doesn’t return. She replies that she would either return to her former life or, more likely, take her own life. When he urges her to accept Yamadori’s proposal, Cio-Cio-San is deeply hurt. She shows Sharpless her son, and asks Sharpless to inform Pinkerton that he is a father—surely this news will make Pinkerton hurry back to her. Sharpless promises her that Pinkerton will know.
Suzuki and Cio-Cio-San hear a cannon shot from the harbor, and they realize it announces the arrival of Pinkerton’s ship, the U.S.S. Abraham Lincoln. They decorate the house in preparation for his return. Night falls, and they settle in to wait for Pinkerton.
At dawn, the drowsy Cio-Cio-San sings a lullaby to her boy and goes to sleep. Pinkerton and Sharpless arrive. Suzuki notices a woman in the garden, and Sharpless tells her that the woman is Kate, Pinkerton’s American wife, who wants to raise Cio-Cio-San’s child as her own. Pinkerton flees, overcome by remorse and unable to confront Cio-Cio-San.
Cio-Cio-San enters and sees Kate in the garden. Cio-Cio-San figures out who Kate is, and Sharpless asks Cio-Cio-San if she will give up the boy. She says she will, if Pinkerton comes in person to ask for his son. Cio-Cio-San makes the ultimate sacrifice on behalf of her child.