Frustrated by the leadership of Tennessean publisher Silliman Evans, Jr., Seigenthaler resigned in 1960 to serve as an administrative assistant to incoming Attorney General Robert F. Kennedy. On April 21, 1961, Seigenthaler was the only other Justice Department figure to witness a meeting between Kennedy and Martin Luther King, Jr.

During the Freedom Rides of 1961, Seigenthaler was sent in his capacity as assistant to Assistant Attorney General for Civil Rights John Doar[8] to be chief negotiator for the government, in its attempts to work with Alabama Governor John Malcolm Patterson. After several days of refusing to return calls, Patterson finally agreed to protect the Riders, but their state trooper escort disappeared as soon as they arrived in Montgomery on May 20, 1961, leaving them unprotected before the waiting white mob.

Seigenthaler was a block away when he rushed to help Susan Wilbur, a Freedom Rider who was being chased by the angry mob. Seigenthaler shoved her into his car and shouted "Get back! I'm with the Federal government" but was hit behind the left ear with a pipe. Knocked unconscious, he was not picked up until police arrived 10 minutes later, with Montgomery Police Commissioner Lester B. Sullivan noting, "We have no intention of standing police guard for a bunch of troublemakers coming into our city."