The new brick building was completed in 1884 under the supervision of its pastor, William R. Pettiford,[6] but in 1908, the city condemned the structure and ordered it to be demolished.

In addition to the main sanctuary, the building houses a basement auditorium, used for meetings and lectures, and several ancillary rooms used for Sunday school and smaller groups.

Du Bois, Mary McLeod Bethune, Paul Robeson and Ralph Bunche all spoke at the church during the first part of the 20th century.

During the civil rights movement of the 1960s, the 16th Street Baptist Church served as an organizational headquarters, site of mass meetings and rallying point for African Americans protesting widespread institutionalized racism in Birmingham, Alabama, and the South.

The ministers Fred Shuttlesworth, who was the chief local organizer, Southern Christian Leadership Conference (SCLC) leader Martin Luther King Jr., and SCLC leader James Bevel, who initiated the Children's Crusade and taught the students nonviolence, were frequent speakers at the church and led the movement.

On Sunday, September 15, 1963, Thomas Blanton, Bobby Frank Cherry and Robert Edward Chambliss, members of the Ku Klux Klan, planted 19 sticks of dynamite outside the basement of the church.

At 10:22 a.m., they exploded, killing four young girls – Addie Mae Collins, Carole Robertson, Cynthia Wesley and Denise McNair.

In 1993, a team of surveyors for the Historic American Buildings Survey executed archival quality measured drawings of the church for the Library of Congress.

Additional funds were sought to handle unexpected problems uncovered during the work and to provide for ongoing physical maintenance.