LaDuke was born in Los Angeles, California to Vincent and Betty LaDuke. Her father was part Anishinaabe (Ojibwe or "Chippewa") from Minnesota. He was an actor with supporting roles in Western movies, an activist, a writer, and at the end of his life, a spiritual guru under the name Sun Bear. Her mother was a Jewish artist, employed as an art professor at Southern Oregon University in Ashland, Oregon.

LaDuke was raised in Ashland, Oregon, Willamette Week | “Winona Laduke” | July 19th, 2006 but after graduating from Harvard in 1982 with a degree in rural economic development, she accepted a job as principal of the high school on the Anishinaabe White Earth Indian Reservation in Minnesota. She then graduated with an M.A. in Community Economic Development from Antioch University. She soon became an activist, involved in the struggle to recover lands promised to the Anishinaabe by an 1867 treaty. She helped the Anishinaabe buy back thousands of acres of ancestral land.

She worked with Women of All Red Nations to publicize the alleged high level of forced sterilization among Native American women.

LaDuke is the author of the novel Last Standing Woman (1997), the non-fiction book All our Relations: Native Struggles for Land and Life (1999), and Recovering the Sacred: the Power of Naming and Claiming (2005), a book about traditional beliefs and practices.

She appeared in the documentary film Anthem, directed by Shainee Gabel and Kristin Hahn. The film was first released in the United States on July 25, 1997. Both directors were awarded by the 1997 Amsterdam International Documentary Film Festival. LaDuke also appeared in the TV documentary The Main Stream, first released on December 17, 2002.

LaDuke was named Woman of the Year by Ms. Magazine in 1997 and won the Reebok Human Rights Award in 1998. She is the founder of the White Earth Land Recovery Project in Minnesota and the Indigenous Women's Network. She is also Executive Director of Honor the Earth, an organization she co-founded with Indigo Girls in 1993, and later sponsored and organized by the Seventh Generation Fund, Indigenous Women's Network and the Indigenous Environmental Network. The Native-led organization's mission is "to create awareness and support for Native environmental issues and to develop needed financial and political resources for the survival of sustainable Native communities. Honor the Earth develops these resources by using music, the arts, the media, and Indigenous wisdom to ask people to recognize our joint dependency on the Earth and be a voice for those not heard." She was also given the Ann Bancroft Award for Women's Leadership Fellowship.

In Sept 2007, LaDuke was inducted into the National Women's Hall of Fame. National Women's Hall of Fame - News & Events

On November 9, 2008 LaDuke's house in Ponsford, Minnesota burned down. Although LaDuke was in Boston when the fire broke out, the four family members at home got out in time. No one was injured, but LaDuke lost all her personal property on the site, including her extensive library and Indigenous art and artifact collection.

LaDuke is the mother of three children, and now a grandmother.

LaDuke Appeared on The Colbert Report June 12, 2008.