Set in Meiji-period Nagasaki. US naval officer Lieutenant Pinkerton, who is about to take a young Japanese bride, is inspecting a house he is leasing from Goro, a marriage broker. He boasts that he can terminate the lease and marriage any time, but the American consul Sharpless, who knows that the bride is serious about the marriage, is worried about his frivolous attitude. The procession of the bride and her friends arrives. Cio-Cio-San, who sings that she is the happiest girl in the world, admits that she is fifteen. Her family was once wealthy but her father was forced to commit suicide, and she now earns her living as a geisha. The ceremony is interrupted by her uncle Bonzo, a Buddhist priest, who denounces her for abandoning her faith. Festivities over, the couple retreat and enjoy the wedding night.

Three years have passed since Pinkerton has left for America. Cio-Cio-San awaits his return, believing his promise that he would come again 'when the robins build their nests'. Sharpless and Goro advise her to remarry, but she declines, as she has had a son by Pinkerton in his absence. Sharpless, who knows that Pinkerton has wed an American wife, leaves in shock. Eventually, Pinkerton's ship arrives in Nagasaki harbor. Cio-Cio-San and Suzuki, her loyal maid, decorate the house with flowers and await his arrival but he doesn't appear. As Cio-Cio-San rests, Sharpless brings Pinkerton and Kate, Pinkerton's new wife, and Suzuki receives them. Pinkerton is full of remorse and runs from the scene. Cio-Cio-San sees Kate instead of Pinkerton and she agrees to give up her child so he could be brought up in America. She takes a final farewell of her child and stabs herself with her father's dagger, choosing "to die with honor rather than live in shame". Pinkerton is heard desperately calling her name.