Lister Hill was born in Montgomery, Alabama on December 29, 1894, the son of one of the South's most distinguished surgeons, Dr. Luther Leonidas Hill. He was named after Dr. Joseph Lister, the father of antiseptic surgery. Following his graduation from Starke University in Montgomery, he entered The University of Alabama at age 16, and graduated four years later with a law degree and a Phi Beta Kappa key. While a student at The University of Alabama, he founded the Student Government Association (SGA) and was its first president, the Jasons Senior Men's Honorary (which the University ceased recognizing in 1976 for its all-male policy, but which still taps fifteen men each spring on the Franklin Mound), and The Machine (the local chapter of Theta Nu Epsilon).

He also studied law at the University of Michigan Law School at Ann Arbor and at Columbia Law School in New York City. He was admitted to the Alabama bar in 1916 and commenced practice in Montgomery, Alabama, and also served as the president of the Montgomery Board of Education from 1917-1922.

Hill was elected August 14, 1923 as Congressman from the Second District of Alabama to fill the vacancy caused by the death of John R. Tyson. He served as Chairman of the House Committee on Military Affairs, and was appointed to the U.S. Senate in 1938 following Senator Hugo Black's appointment to the United States Supreme Court. Hill was subsequently elected to the Senate as a Democrat on April 26, 1938, to fill the vacancy caused by the resignation of Sen. Bibb Graves for the term ending January 3, 1939; he was reelected in 1938, 1944, 1950, 1956, and again in 1962. He retired in January 1969.

As a U.S. Senator, Hill was known as a moderate. He distinguished himself in a number of fields, but was best-known for his landmark legislation in the field of public health. Perhaps the best-known legislation which bears his name is the Hospital and Health Center Construction Act of 1946, better known as the Hill-Burton Act. He also sponsored the Hill-Harris Act of 1963, providing for assistance in constructing facilities for the mentally retarded and mentally ill. Additionally, he was recognized as the most instrumental man in Congress in gaining greatly increased support for medical research at the nation's medical schools and other research institution.

He also sponsored other important legislation, including the TVA Act, the Rural Telephone Act, the Rural Housing Act, the Vocational Education Act, and the National Defense Education Act.

Hill signed "The Southern Manifesto" condemning the Supreme Court decision in Brown vs Board of Education ordering school desegregation (although he remained a close friend of Supreme Court Justice and fellow Alabaman Hugo Black, who voted for Brown).

However, Lister Hill was as much a national figure as a representative of Alabama and the South. During his long years in the Congress, he would, from time to time, break with his southern colleagues to follow his own conscience. For example, in opposition to most southerners in the Congress, he favored federal control of offshore oil with revenue to be earmarked for education.

Hill was the Senate Majority Whip from 1941-1947. He was Chairman of the Senate Labor and Public Welfare Committee, which handled important legislation on veterans education, health, hospitals, libraries, and labor-management relations. He was also a ranking member of the Senate Appropriations Committee, and a member of the Senate Democratic Policy Committee.

He received honorary degrees from 13 colleges and universities, including the University of Alabama and Auburn University. He was a Methodist, a Freemason, a United States Army veteran of World War I with the Seventeenth and Seventy-first United States Infantry Regiments, and a member of the American Legion.

Hill died in Montgomery, Alabama on December 21, 1984, and is interred in Greenwood Cemetery.