O'Keeffe grew up and attended schools in her hometown of Sun Prairie, Wisconsin, and, from 1902, in Williamsburg, Virginia. Determined from an early age to be a painter, she studied at the Art Institute of Chicago (1904–05) and the Art Students League of New York (1907–08), and afterward she supported herself by doing commercial art. She then taught art at various schools and colleges in Texas and other Southern states from 1912 to 1916, and in the latter year her drawings were discovered and exhibited by the American photographer Alfred Stieglitz (Stieglitz, Alfred). Stieglitz praised and promoted her work, and the two artists began a lifelong relationship, marrying in 1924. The hundreds of photographs Stieglitz took of her form a notable and extended portrait series. O'Keeffe moved to New York City after meeting Stieglitz; she later spent periods in New Mexico, to which she moved after her husband's death in 1946.

O'Keeffe painted her best-known works in the 1920s, '30s, and '40s, but she remained an active painter into the '80s. Her later works frequently celebrate the clear skies and desert landscapes of New Mexico. A retrospective exhibition of her art held at the Whitney Museum of American Art in 1970 assured her reputation as one of the most original and important artists in modern American painting.

Her autobiography, Georgia O'Keeffe, was published in 1976.