In 1960, Burke Marshall was an attorney in Washington, D.C. When President John F. Kennedy took office in 1961, he appointed his brother Robert Kennedy United States Attorney General. Marshall was appointed an assistant attorney general, and led a team of lawyers who made up the new Civil Rights Division within the Department of Justice. His tenure in the Justice Department coincided with some of the most tumultuous years of the Civil Rights movement. Marshall was an aggressive advocate for racial equality and a leading figure in the federal government's efforts to desegregate the South.

Marshall mediated conflicts between civil rights advocates and southern white officials. After the 1961 Freedom Rides and the ensuing violence, Marshall helped implement the Interstate Commerce Commission's desegregation of interstate travel. In 1962, he represented the Kennedy administration in its confrontation with state and educational officials who refused to admit a black student named James Meredith to the University of Mississippi. Both events sparked controversy, white resistance, and violence that required the National Guard to restore order. Marshall later recalled the intensity of the situation: "There was a period in which I really thought we [federal government soldiers] were going to have to shoot our way into campus."

Marshall was instrumental in defusing racial tensions between protesters and city officials in Birmingham, Alabama, in the spring of 1963. That April, civil rights activists started a series of sit-ins to protest public eating establishments that refused to serve African Americans. It was during these demonstrations that the Reverend Martin Luther King Jr. was arrested and jailed; he wrote his famous "Letter from Birmingham Jail" during his incarceration.

On May 2, African American elementary, high school, and college students in Birmingham began a series of nonviolent protest marches. Public Safety Commissioner Eugene "Bull" Connor ordered law enforcement officials to use police dogs and fire hoses to disperse the marchers, creating a crisis that was covered on national television and broadcast around the world. More than 2,000 students were arrested and jailed in what became known as the Children's Crusade. During the crisis, Marshall traveled to Birmingham "to try to get in the middle of it and see if it couldn't be resolved." While in Birmingham, Marshall, as one historian explains, "got the leaders of the white community to at least listen to African Americans' complaints and he arranged a truce that eased a dangerous situation and opened the way for gradual desegregation."

At the Department of Justice, Marshall also participated in the writing and implementation of the Civil Rights Act of 1964, which prohibited racial discrimination in public places, effectively outlawing segregation.