On a terrace above Nagasaki harbour, US Navy Lieutenant B F Pinkerton inspects the house he has leased from a marriage broker, Goro, who has procured for him a geisha wife known as Madama Butterfly (Cio-Cio-San). To the American Consul, Sharpless, who arrives breathless from climbing the hill, Pinkerton describes his carefree philosophy of a sailor roaming the world in search of pleasure. For the moment, he is enchanted with the fragile Cio-Cio-San and intends to undergo a marriage ceremony with her – a 999-year contract, but subject to monthly renewal. When Sharpless warns that the girl may not take her vows so lightly, the lieutenant brushes aside such scruples, adding that he will one day take a 'real' American wife.
Cio-Cio-San is heard in the distance joyously singing of her wedding day. After she has entered, surrounded by her friends, she tells Pinkerton how, when her family fell on hard times, she had to earn her living as a geisha. Soon her relatives arrive and noisily express their opinions of the marriage. In a quiet moment, Cio-Cio-San shows the bridegroom her little store of possessions, one of which she hides from public view. Goro explains that it is a sheathed knife which the Mikado sent to Butterfly's father, with the 'invitation' to commit hara-kiri – which he obeyed. Butterfly confesses to Pinkerton that she, on the previous evening, secretly went to the Mission and adopted the religion of her new husband.
The wedding ceremony completed, the guests toast the couple. Suddenly Cio-Cio-San's uncle, a priest, bursts upon the scene, cursing the girl for having renounced her ancestors' religion. Pinkerton angrily orders priest and family to leave.
Alone with his bride, he dries her tears in the moonlit garden, where they discover the depths of their love.
Three years later, Cio-Cio-San still waits for her husband's return. Suzuki prays to her gods for aid. The maid shows Cio-Cio-San how little money is left but is told to have faith: one fine day Pinkerton's ship will appear on the horizon.
Sharpless is announced. He has not seen her again since the wedding, and Butterfly receives him with joy. He has come with a letter from Pinkerton asking him tactfully to inform Butterfly of his marriage with an American woman, but his attempts to tell her the contents of the letter are frustrated by her constant questions about Pinkerton. Had Pinkerton not said that he would return 'in the season when the robins are nesting?' In Japan, she remarks, 'the robins have already nested three times, but perhaps in America these birds behave differently?' 'I never studied ornithology', replies Sharpless.
Goro, who has been lurking outside, brings in a suitor for her hand. The girl dismisses the wealthy Prince Yamadori, insisting that her American husband has not deserted her. When they are alone, Sharpless again starts to read her the letter and suggests as tactfully as he can that Pinkerton may never return. Cio-Cio-San proudly shows him her child, insisting that as soon as Pinkerton knows of his son he will surely come back, though if he does not she would rather die than return to her former life. Moved by her devotion and lacking the heart to tell her of the lieutenant's marriage, Sharpless leaves.
Cio-Cio-San, on the point of despair, hears a cannon report; and watches Pinkerton's ship entering the harbour. Delirious with joy, she orders Suzuki to help her strew the house with flower petals. Then, as night falls, Cio-Cio-San, Suzuki and the child begin their vigil, awaiting Pinkerton's arrival.
As dawn breaks, Suzuki insists that Cio-Cio-San rests. Humming a lullaby to her child, she carries him to another room.
Knocking is heard: it is Pinkerton and Sharpless, with Pinkerton's wife, Kate, remaining discreetly outside. They have come, they explain to the startled Suzuki, so early in the morning in the hope of finding her alone and of enlisting her support in persuading Butterfly to accept Kate's offer to adopt the child. Pinkerton, overcome with remorse, bids an anguished farewell to the scene of his former happiness and rushes away.
Meanwhile Suzuki has gone into the garden to speak to Kate and, moved by her sincerity, she promises to convey to her mistress her offer to adopt the child. Butterfly rushes into the room in joyful expectation to find Pinkerton, but is taken aback when she sees only Sharpless and a foreign lady. She takes only a moment to guess the truth. She agrees to give up her child if the father will return for him. Then, she takes forth the dagger with which her father committed suicide, choosing to die with honour rather than live in disgrace. Just as she raises the blade, Suzuki pushes the child into the room. Tearfully she bids him a last farewell. With solemn ritual, she stabs herself as Pinkerton's anxious cries 'Butterfly! Butterfly!' are heard from outside.