The Anacostia Museum grew out of an idea that was first discussed at a conference on museums and education sponsored by the Smithsonian in August 1966. Soon thereafter, Smithsonian Secretary S. Dillon Ripley formed a committee to plan "an experimental store-front museum" in a Washington, D.C., neighborhood. In March 1967, the Smithsonian secured the Carver Theater in Anacostia as the site for the project.

Anacostia community leaders formed an advisory council to guide the venture and build local support. In June the Institution appointed John R. Kinard as Director of the Museum, a position he held until his death in 1989. Smithsonian staff cooperated with local citizens to convert the theater into an exhibition space, and to select objects for display. The theater was renamed the Anacostia Neighborhood Museum, and opened to the public on September 15, 1967.

The Museum relied largely on special grants for support until 1970, when it became a line item in the Institution's federal budget. Charles Blitzer, Director of the Office of Education and Training, was the Institution's chief liaison with the Museum until those duties were transferred to William W. Warner, Acting Assistant Secretary for Public Service, in November 1968. Assistant Secretary for History and Art John E. Reinhardt took over administration of the Museum in 1983.

In October 1974, the Exhibit Branch of the Museum moved into the new Exhibits Design and Production Laboratory in Fort Stanton Park. This facility served as the core for a larger museum building that was completed in 1987. The new structure was large enough to accommodate all the functions of the Museum in one location for the first time.

The Museum implemented an acquisition program in 1977, and first used original artifacts in the 1979 exhibition Out of Africa: From West African Kingdoms to Colonization. Other exhibitions have examined such subjects as urban problems, the history of Anacostia, African American art and heritage, and African culture. In April 1987 the Museum had its name changed to the Anacostia Museum. This reflected the Museum's increased mandate to examine, preserve, and interpret African American history and culture, not only locally and regionally, but nationally and internationally as well.