Born in Missoula, Montana on June 11, 1880, Rankin was the first of seven children of Canadian immigrant John Rankin, a rancher and lumber merchant, and Olive Rankin (née Pickering), a former school teacher originally from New England. She attended the University of Montana and graduated in 1902 with a bachelor of science degree in biology.

In 1908, she migrated to New York City, where she started a career as a social worker. She later moved to Seattle, Washington, and then enrolled at the University of Washington, where she joined the incipient suffrage cause. She was instrumental in the cause's efforts to enable women to vote in Montana, and women gained the vote in Montana in 1914. On November 7, 1916 she was elected to the House of Representatives as a Republican from Montana, becoming the first female member of Congress. The Nineteenth Amendment (which gave women the right to vote everywhere in the United States) was not ratified until 1920; therefore, during Rankin's first term in Congress (1917–1919), many women throughout the country did not have the right to vote, though they did in her home state of Montana.

On April 6, 1917, only four days into her term, the House voted on the resolution to enter World War I. Rankin cast one of 50 votes against the resolution, earning her immediate vilification by the press. Suffrage groups canceled her speaking engagements. Despite her vote against entering the war, she devoted herself to selling Liberty Bonds and voted for the military draft.

In 1918, she ran an unsuccessful campaign for the Republican nomination to represent Montana in the United States Senate. She then ran an independent candidacy, which also failed. Her term as Representative ended early in 1919. For the next two decades, she worked as a lobbyist in Washington, D.C. for various causes.

In 1918, and again in 1919, she introduced legislation to provide state and federal funds for health clinics, midwife education, and visiting nurse programs in an effort to reduce the nation's infant mortality. While serving as a field secretary for the National Consumers' League, she campaigned for legislation to promote maternal and child health care. As a lobbyist, Rankin argued for passage of the Sheppard-Towner Act, an infant and maternal health bill which was the first federal social welfare program created explicitly for women and children. The legislation, however, was not enacted until 1921 and was repealed just eight years later. She was founding Vice-President of the American Civil Liberties Union and a founding member of the Women's International League for Peace and Freedom.

In 1940, Rankin was again elected to Congress, this time on an anti-war platform. Following the attack on Pearl Harbor, she once again voted against entering a World War, the only member of Congress to do so, saying "As a woman, I can't go to war and I refuse to send anyone else. It is not necessary. I vote NO." Montana Republican leaders demanded that Rankin change her vote, but she refused. However she did not vote against declaring war on Germany and Italy following their declaration of war on the U.S. Instead, she voted merely Present. By 1942, Rankin's antiwar stance had become so unpopular that she did not seek re-election. During the remainder of her life, she traveled to India seven times and was a devotee of Gandhian principles of non-violence and self-determination.

Rankin was actively opposed to U.S. military action in the Vietnam War. In 1968 she led a protest demonstration of thousands of women in Washington, D.C. The following year she participated in other antiwar marches in Georgia and South Carolina.
Rankin died in Carmel, California at the age of 92 from natural causes. Rankin bequeathed her property in Watkinsville, Georgia to help "mature, unemployed women workers." This was the seed money for the Jeannette Rankin Foundation, a 501(c)(3) organization that gives educational scholarships annually to low income women all across the United States. The organization has built capacity since its single $500 scholarship in 1978 to the eighty $2000 scholarships it is awarding in 2007. In 1985, a statue of her was placed in the United States Capitol's Statuary Hall. A play based on the life of Rankin entitled A Single Woman was produced in 2004, and a film of the same name was made in 2008.