Her father, Larry Walker, was an artist and chair of the art department at the University of the Pacific in Stockton. She showed promise as an artist from a young age, but it was not until the family moved to Georgia when she was 13 that she began to focus on issues of race. Walker received a bachelor's degree (1991) from the Atlanta College of Art and a master's degree (1994) from the Rhode Island School of Design, where she began working in the silhouette form while exploring themes of slavery, violence, and sex found in sources such as books, films, and cartoons.

Later in 1994 Walker's work appeared in a new-talent show at the Drawing Center in New York. Her contribution was a 50-foot (15-metre) mural of life-size silhouettes depicting a set of disturbing scenes set in the antebellum South. The piece was titled Gone, a Historical Romance of a Civil War as It Occurred Between the Dusky Thighs of One Negress and Her Heart. That work and subsequent others, such as a series of watercolours titled Negress Notes (Brown Follies) (1996–97), caused a stir. Some African American artists, particularly those of the civil rights era, deplored her use of racist caricatures. Walker made it clear that her intent as an artist was not to create pleasing images or to raise questions with easy answers.

In 1997, at age 27, Walker received a John D. and Catherine T. MacArthur Foundation “genius grant.” Her work was exhibited in galleries and museums worldwide, and she served as the U.S. representative to the 2002 São Paulo Biennial. She was also on the faculty of the School of the Arts at Columbia University in New York City. In 2006 the Metropolitan Museum of Art in New York City featured her exhibition titled “After the Deluge,” which was inspired in part by the devastation wreaked the previous year by Hurricane Katrina in New Orleans. The exhibition juxtaposed pieces from the museum's own collection—many of which depicted black figures or images demonstrating the terrific power of water—with some of her own works. The intermingled disparate images created an amalgam of new meaning fraught with a discomfiting ambiguity characteristic of much of Walker's output.