Eastland was born in Doddsville. In 1905 he moved with his parents to Forest where he attended public schools. A lawyer in rural Mississippi, he served two terms in the state House of Representatives from 1928 to 1932. He was first appointed to the Senate in 1941 following the death of Senator Pat Harrison, but did not run in the special election for the seat later in the year; it was won by 2nd District Congressman Wall Doxey. In 1942, Eastland was one of three candidates who challenged Doxey for a full term. Even though Doxey had the support of President Franklin Delano Roosevelt, Eastland defeated him in the Democratic primary, which at that time was tantamount to election in Mississippi. He was reelected five times, only facing substantive Republican opposition twice. In 1966, 4th District Congressman Prentiss Walker, the first Republican to represent Mississippi at the federal level in over 80 years, ran against him. However, as is usually the case with Congressmen who run for the Senate after only one term, Walker was badly defeated. In 1972, Eastland was reelected with 58f the vote in his closest contest ever. His Republican opponent, Gil Carmichael, was undoubtedly aided by President Richard Nixon's landslide reelection in 49 states, including 78f Mississippi's popular vote.

An open, unashamed, tough-talking and ruthless racist and segregationist, Eastland was a violent opponent of the American Civil Rights Movement. When the Supreme Court issued its decision in the landmark case Brown v. Board of Education of Topeka, Kansas 347 US 483 (1954), Eastland denounced it, saying:

"On May 17, 1954, the Constitution of the United States was destroyed because of the Supreme Court's decision. You are not obliged to obey the decisions of any court which are plainly fraudulent sociological considerations." When three civil rights workers Mickey Schwerner, James Chaney, and Andrew Goodman went missing in Mississippi on June 21, 1964, he reportedly told President Lyndon Johnson that the incident was a hoax and there was no Ku Klux Klan in the state, surmising that the three had gone to Chicago. As such, he was portrayed in the Hollywood version of the incident, Mississippi Burning. Eastland served as a director of the infamous Pioneer Fund, a foundation dedicated to "improving the race." (Eastland would some years later stare coldly down a committee table at Senator Jacob Javits of New York, who was Jewish and say, "I don't like you-or your kind.") Eastland, along with Senators Robert Byrd, John McClellan, Olin D. Johnston, Sam Ervin and Strom Thurmond made unsuccessful attempts to block Thurgood Marshall's confirmation to the Federal Court of Appeals and the Supreme Court.

Eastland was appointed as chairman of the Senate Judiciary Committee in 1956 and held that post until his retirement. Ironically, his committee considered the Civil Rights Act of 1964, which Eastland bitterly opposed. Its passage caused Eastland and most other prominent Mississippi Democrats to openly support Barry Goldwater's presidential bid that year. Although Goldwater was heavily defeated by incumbent Lyndon Johnson, he carried Mississippi with an unheard-of 87f the popular vote due to his opposition to federal civil rights legislation such as the Civil Rights Act of 1964.

During his last Senate term, he served as president pro tem of the Senate since he was the longest-serving Democrat in the Senate.

Eastland was an ally of Joseph McCarthy and served on the Committee investigating many Americans' connections to the Communist Party. Even after McCarthy was discredited, Eastland tried to press the issue. Using his power as chairman of the Internal Security Subcommittee, he subpoenaed a number of employees of The New York Times, which was at the time taking a strong position on its editorial page that Mississippi should adhere to the Brown decision. The Times was not intimidated. Its January 5, 1956 editorial read in part:

"Our faith is strong that long after Senator Eastland and his present subcommittee are gone, long after segregation has lost its final battle in the South, long after all that was known as McCarthyism is a dim, unwelcome memory, long after the last Congressional committee has learned that it cannot tamper successfully with a free press, The New York Times will be speaking for (those) who make it, and only for (those) who make it, and speaking, without fear or favor, the truth as it sees it." In his last years in the Senate he avoided associating himself with racist stands in the face of increasing black political power in Mississippi. As a result he decided not to seek reelection in 1978. Due to an independent black candidate siphoning off votes from the Democratic candidate, Republican 4th District Congressman Thad Cochran won the race to succeed him. Eastland resigned two days after Christmas, giving Cochran a leg up in seniority. Eastland died on February 19, 1986.

The law library at Ole Miss is named after Eastland. This has caused a good deal of controversy in Mississippi given Eastland's unabashed racism.