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Japan, early twentieth century. On a flowering terrace above Nagasaki harbor, U.S. Navy Lieutenant B. F. Pinkerton inspects the house he has leased from a marriage broker, Goro, who has just procured him three servants and a geisha wife, Cio-Cio-San, known as Madama Butterfly. To the American consul, Sharpless, who arrives breathless from climbing the hill, Pinkerton describes the carefree philosophy of a sailor roaming the world in search of pleasure. At the moment, he is enchanted with the fragile Cio-Cio-San, but his 999-year marriage contract contains a monthly renewal option. When Sharpless warns that the girl may not take her vows so lightly, Pinkerton brushes aside such scruples, saying he will one day marry a 'real' American wife. Cio-Cio-San is heard in the distance joyously singing of her wedding. Entering surrounded by friends, she tells Pinkerton how, when her family fell on hard times, she had to earn her living as a geisha. Her relatives bustle in, noisily expressing their opinions on the marriage. In a quiet moment, Cio-Cio-San shows her bridegroom her few earthly treasures and tells him of her intention to embrace his Christian faith. The Imperial Commissioner performs the wedding ceremony, and the guests toast the couple. The celebration is interrupted by Cio-Cio-San's uncle, a Buddhist priest, who bursts in, cursing the girl for having renounced her ancestors' religion. Pinkerton angrily sends the guests away. Alone with Cio-Cio-San in the moonlit garden, he dries her tears, and she joins him in singing of their love.
Three years later, Cio-Cio-San waits for her husband's return. As Suzuki prays to her gods for aid, her mistress stands by the doorway with her eyes fixed on the harbor. When the maid shows her how little money is left, Cio-Cio-San urges her to have faith; one fine day Pinkerton's ship will appear on the horizon. Sharpless brings a letter from the lieutenant, but before he can read it to Cio-Cio-San, Goro comes with a suitor, the wealthy Prince Yamadori. The girl dismisses both marriage broker and prince, insisting her American husband has not deserted her. When they are alone, Sharpless again starts to read the letter and suggests Pinkerton may not return. Cio-Cio-San proudly carries forth her child, Dolore (Trouble), saying that as soon as Pinkerton knows he has a son he surely will come back; if he does not, she would rather die than return to her former life. Moved by her devotion, Sharpless leaves, without having revealed the full contents of the letter. Cio-Cio-San, on the point of despair, hears a cannon report; seizing a spyglass, she discovers Pinkerton's ship entering the harbor. Now delirious with joy, she orders Suzuki to help her fill the house with flowers. As night falls, Cio-Cio-San, Suzuki and the child begin their vigil.
As dawn breaks, Suzuki insists that Cio-Cio-San rest. Humming a lullaby to her child, she carries him to another room. Before long, Sharpless enters with Pinkerton, followed by Kate, his new wife. When Suzuki realizes who the American woman is, she collapses in despair but agrees to aid in breaking the news to her mistress. Pinkerton, seized with remorse, bids an anguished farewell to the scene of his former happiness, then rushes away. When Cio-Cio-San comes forth expecting to find him, she finds Kate instead. Guessing the truth, the shattered Cio-Cio-San agrees to give up her child if his father will return for him. Then, sending even Suzuki away, she takes out the dagger with which her father committed suicide and bows before a statue of Buddha, choosing to die with honor rather than live in disgrace. As she raises the blade, Suzuki pushes the child into the room. Sobbing farewell, Cio-Cio-San sends him into the garden to play, then stabs herself. As she dies, Pinkerton is heard calling her name.