Gregory began performing comedy in the mid-1950s while serving in the army. (see Blacks in the American Military). Drafted in 1954 while attending Southern Illinois University at Carbondale on a track scholarship, Gregory briefly returned to the university after his discharge in 1956, but left without a degree because he felt that the university "didn't want me to study, they wanted me to run." In the hopes of performing comedy professionally, he moved to Chicago, where he became part of a new generation of black comedians that included Nipsey Russell, Bill Cosby, and Godfrey Cambridge. These comedians broke with the minstrel tradition, which presented stereotypical black characters. Gregory, whose style was detached, ironic, and satirical, came to be called the "Black Mort Sahl" after the popular white social satirist. He drew on current events, especially the racial issues, for much of his material: "Segregation is not all bad. Have you ever heard of a collision where the people in the back of the bus got hurt?"

From an early age, Gregory demonstrated a strong sense of social justice. While a student at Sumner High School in St. Louis he led a march protesting segregated schools. Later, inspired by the work of leaders such as Dr. Martin Luther King Jr. and organizations such as the Student Nonviolent Coordinating Committee (SNCC), Gregory took part in the Civil Rights Movement and used his celebrity status to draw attention to such issues as segregation and disfranchisement. When local Mississippi governments stopped distributing federal food surpluses to poor blacks in areas where SNCC was encouraging voter registration, Gregory chartered a plane to bring in seven tons of food. He participated in SNCC's voter registration drives and in sit-ins to protest segregation, most notably at a restaurant franchise in downtown Atlanta, Georgia. Only later did Gregory disclose that he held stock in the chain.

Through the 1960s, Gregory spent more time on social issues and less time on performing. He participated in marches and parades to support a range of causes, including opposition to the Vietnam War, world hunger, and drug abuse. In addition, Gregory fasted in protest more than 60 times, once in Iran, where he fasted and prayed in an effort to urge the Ayatollah Khomeini to release American embassy staff who had been taken hostage. The Iranian refusal to release the hostages did not decrease the depth of Gregory's commitment; he weighed only 97 lbs when he left Iran.

Gregory demonstrated his commitment to confronting the entrenched political powers by opposing Richard J. Daley in Chicago's 1966 mayoral election. He ran for president in 1968 as a write-in candidate for the Freedom and Peace Party, a splinter group of the Peace and Freedom Party and received 1.5 million votes. Democratic candidate Hubert Humphrey lost the election to Republican Richard Nixon by 510,000 votes, and many believe Humphrey would have won had Gregory not run. After the assassinations of King, President John F. Kennedy, and Robert Kennedy, Gregory became increasingly convinced of the existence of political conspiracies. Gregory wrote books such as Code Name Zoro: The Murder of Martin Luther King Jr. (1971) with Mark Lane, a conspiracy theorist whose ideas Gregory shared and espoused in numerous lectures.

Gregory's activism continued into the 1990s. In response to published allegations that the Central Intelligence Agency (CIA) had supplied cocaine to predominantly African American areas in Los Angeles, thus spurring the crack epidemic, Gregory protested at CIA headquarters and was arrested. In 1992 he began a program called "Campaign for Human Dignity" to fight crime in St. Louis neighborhoods.

In 1973, the year he released his comedy album Caught in the Act, Gregory moved with his family to Plymouth, Massachusetts, where he developed an interest in vegetarianism and became a nutritional consultant. In 1984 he founded Health Enterprises, Inc., a company that distributed weight loss products. In 1987 Gregory introduced the Slim-Safe Bahamian Diet, a powdered diet mix, which was immensely profitable. Economic losses caused in part by conflicts with his business partners led to his eviction from his home in 1992. Gregory remained active, however, and in 1996 returned to the stage in his critically acclaimed one-man show, Dick Gregory Live!.