John Seigenthaler, the oldest of eight children, was born in Nashville, Tennessee, on the 27th July, 1927. He attended Father Ryan High School and served in the U.S. Air Force after the Second World War.

In 1949 Seigenthaler was employed by The Tennessean. During this period he took courses in sociology and literature at Peabody College, Vanderbilt. He also attended the American Press Institute at Columbia University. He was a talented journalist and won the National Headliner Award for his story about Thomas C. Buntin, a wealthy Nashville business owner who had disappeared in September 1931, but was discovered by Seigenthaler living in Orange, Texas.

In July 1957, Seigenthaler began to investigate corruption within the local branch of the Teamsters. He also looked into the criminal activities of Dave Beck and Jimmy Hoffa. His articles led to the impeachment trial of Chattanooga Criminal Court Judge Ralston Schoolfield. In 1958 Seigenthaler became an assistant city editor and special assignment reporter. Seigenthaler was a supporter of John F. Kennedy in the 1960 Presidential Election and after his victory he was appointed as administrative assistant to Attorney General Robert F. Kennedy.

The civil rights group, the Congress on Racial Equality (CORE) began to organize Freedom Rides in an attempt to bring an end to segregation in transport. After three days of training in non-violent techniques, black and white volunteers sat next to each other as they travelled through the Deep South.

James Farmer, national director of CORE, and thirteen volunteers left Washington on 4th May, 1961, for Georgia, Alabama and Mississippi. Governor James Patterson commented that: "The people of Alabama are so enraged that I cannot guarantee protection for this bunch of rabble-rousers." Patterson, who had been elected with the support of the Ku Klux Klan added that integration would come to Alabama only "over my dead body."

The Freedom Riders were split between two buses. They travelled in integrated seating and visited "white only" restaurants. When they reached Anniston on 14th May the Freedom Riders were attacked by men armed with clubs, bricks, iron pipes and knives. One of the buses was fire-bombed and the mob held the doors shut, intent on burning the riders to death.

The surviving bus travelled to Birmingham, Alabama. A meeting of Student Nonviolent Coordinating Committee (SNCC) at Fisk University in Nashville, Tennessee decided to send reinforcements. This included John Lewis, James Zwerg, and eleven others including two white women. The volunteers realized their mission was extremely dangerous. Zwerg later recalled: "My faith was never so strong as during that time. I knew I was doing what I should be doing." Zwerg wrote a letter to his parents that stated that he would probably be dead by the time they received it.

During the Freedom Riders campaign the Attorney General, Robert Kennedy was phoning Jim Eastland “seven or eight or twelve times each day, about what was going to happen when they got to Mississippi and what needed to be done. That was finally decided was that there wouldn’t be any violence: as they came over the border, they’d lock them all up.” When they were arrested Kennedy issued a statement as Attorney General criticizing the activities of the Freedom Riders. Kennedy sent John Seigenthaler to negotiate with Governor James Patterson of Alabama. Harris Wofford, the president's Special Assistant for Civil Rights, later pointed out: "Seigenthaler arrived in time to escort the first group of wounded and shaken riders from the bus terminal to the airport, and flew with them to safety in New Orleans."

The Freedom Riders now traveled onto Montgomery. One of the passengers, James Zwerg, later recalled: "As we were going from Birmingham to Montgomery, we'd look out the windows and we were kind of overwhelmed with the show of force - police cars with sub-machine guns attached to the backseats, planes going overhead... We had a real entourage accompanying us. Then, as we hit the city limits, it all just disappeared. As we pulled into the bus station a squad car pulled out - a police squad car. The police later said they knew nothing about our coming, and they did not arrive until after 20 minutes of beatings had taken place. Later we discovered that the instigator of the violence was a police sergeant who took a day off and was a member of the Klan. They knew we were coming. It was a set-up."

The passangers were attacked by a large mob. They were dragged from the bus and beaten by men with baseball bats and lead piping. Taylor Branch, the author of Parting the Waters: America in the King Years, 1954-63 (1988) wrote: "One of the men grabbed Zwerg's suitcase and smashed him in the face with it. Others slugged him to the ground, and when he was dazed beyond resistance, one man pinned Zwerg's head between his knees so that the others could take turns hitting him. As they steadily knocked out his teeth, and his face and chest were streaming blood, a few adults on the perimeter put their children on their shoulders to view the carnage." Zwerg later argued: "There was noting particularly heroic in what I did. If you want to talk about heroism, consider the black man who probably saved my life. This man in coveralls, just off of work, happened to walk by as my beating was going on and said 'Stop beating that kid. If you want to beat someone, beat me.' And they did. He was still unconscious when I left the hospital. I don't know if he lived or died."

Some of the Freedom Riders, including seven women, ran for safety. The women approached an African-American taxicab driver and asked him to take them to the First Baptist Church. However, he was unwilling to violate Jim Crow restrictions by taking any white women. He agreed to take the five African-Americans, but the two white women, Susan Wilbur and Susan Hermann, were left on the curb. They were then attacked by the white mob.

John Seigenthaler, who was driving past, stopped and got the two women in his car. According to Raymond Arsenault, the author of Freedom Riders (2006): "Suddenly, two rough-looking men dressed in overalls blocked his path to the car door, demanding to know who the hell he was. Seigenthaler replied that he was a federal agent and that they had better not challenge his authority. Before he could say any more, a third man struck him in the back of the head with a pipe. Unconscious, he fell to the pavement, where he was kicked in the ribs by other members of the mob. Pushed under the rear bumper of the car, his battered and motionless body remained there until discovered by a reporter twenty-five minutes later."

Harris Wofford, the president's Special Assistant for Civil Rights, pointed out: "Seigenthaler went to the defense of a girl being beaten and was clubbed to the ground; he was kicked while he lay there unconscious for nearly half an hour. Again FBI agents present did nothing, except take notes." Robert F. Kennedy later reported: "I talked to John Seigenthaler in the hospital and said that I thought it was very helpful for the Negro vote, and that I appreciated what he had done."

In March 1962, Seigenthaler was appointed as editor of The Tennessean. He continued his campaign against Jimmy Hoffa. As a result Hoffa's lawyers attempted to move his jury tampering trial from Nashville. Seigenthaler admitted he personally wanted Hoffa convicted and the trial was moved to Chattanooga, but Hoffa was still convicted in 1964 after a 45-day trial.

Seigenthaler was given leave from his newspaper to work on Robert Kennedy's 1968 presidential campaign. Kennedy was shot by an assassin and died on 6th June, 1968. Seigenthaler serve as one of the pallbearers at his funeral, and later co-edited the book An Honorable Profession: A Tribute to Robert F. Kennedy (1993) with Pierre Salinger.

On 8th February, 1973, Seigenthaler was promoted to publisher of The Tennessean. He worked closely with Al Gore on investigative stories about Nashville City Council corruption. On 5th May, 1976, Seigenthaler dismissed Jacque Srouji, a copy editor at the newspaper, after finding that she had served as an informant for the Federal Bureau of Investigation (FBI). At the time she was writing a book critical of Karen Silkwood. Seigenthaler tried for a year to get his own FBI dossier, and finally received some highly expurgated material including these words: "Allegations of Seigenthaler having illicit relations with young girls, which information source obtained from an unnamed source."

In May 1982, Seigenthaler also became editorial director of USA Today. In 1986, Middle Tennessee State University established the John Seigenthaler Chair of Excellence in First Amendment Studies, honoring Seigenthaler's "lifelong commitment to free expression values". He resigned from most newspapers in December 1991. Later that month he founded the First Amendment Center at Vanderbilt University.

In May 2005, an anonymous user, created a Wikipedia article about Seigenthaler which claimed "was thought to have been directly involved in the Kennedy assassinations of both John, and his brother, Bobby." After investigative work by Daniel Brandt, the culprit was identified as Brian Chase, a manager at a small delivery service in Nashville.