Joseph Lenoir Chambers Jr. was born in Charlotte, North Carolina, to Joseph Lenoir Chambers Sr. and Grace Singleton Dewey Chambers. He received his early education in Charlotte's public schools and Woodberry Forest (preparatory) School in Virginia. While attending the University of North Carolina, he excelled at academics, played varsity sports, and edited the campus newspaper. Following his graduation in 1914, he taught at Woodberry Forest for two years, then enrolled in Columbia University's School of Journalism. During World War I (1914–1918), he served as a first lieutenant in the U.S. Army and briefly commanded a company in combat in France. At war's end Chambers returned to the University of North Carolina to direct its news bureau.

Soon after joining the Greensboro Daily News in 1921, he was made its associate editor and worked closely with Earle Godbey, the independent-minded paper's widely respected editor. In 1928 Chambers married the newspaper's society editor, Roberta Burwell Strudwick, who had a son, Robert Strudwick Glenn, from a previous marriage. The couple's daughter, Elisabeth Lacy, was born nine years later.

In 1929 Chambers became the associate editor of the Norfolk Virginian-Pilot, where he worked closely with its distinguished editor, Louis Isaac Jaffé, who had just become Virginia's first Pulitzer Prize-winner for his antilynching advocacy. In 1944 Chambers became editor of Norfolk 's afternoon newspaper, the Ledger-Dispatch. When Jaffé—by this time a close friend and colleague—died in 1950, Chambers became editor of the Pilot. This proved a difficult move for Chambers because he always thought of the editor's office as Jaffé's; Chambers often joked he knew Jaffé better than he knew his own wife.

Chambers, whom friends described as "courtly," enjoyed an impeccable reputation as a "Son of the South" and was not a racial activist. Nevertheless he did become one of a small group of white southern editors who urged compliance with Brown v. Board of Education of Topeka, Kansas, the Supreme Court of the United States 1954 landmark desegregation mandate. For five years after the decision, he conducted an unrelenting editorial campaign opposing the Massive Resistance directed by the political machine led by Harry F. Byrd Sr., Virginia's powerful senior senator. Chambers's editorials helped prepare the public for eventual desegregation and peaceful integration of Virginia schools, which was finally achieved only after the U.S. Supreme Court's 1968 ruling in Green et al. v. County School Board of New Kent County et al.

Chambers's journalistic advocacy reached a high point in the fall of 1958 and winter of 1959, after Governor James Lindsay Almond Jr. closed Norfolk's white secondary schools. The elderly editor demanded their reopening. Chambers provided leadership for pro-school forces and, early in 1959, state and federal courts reopened the city's schools. That same year, Chambers's critically acclaimed two-volume biography of Confederate general Stonewall Jackson was published, and in 1960 he won the Pulitzer Prize for Distinguished Editorial Writing for his school advocacy campaign.

After retiring from the Pilot in 1961, Chambers continued to write (his Salt Water and Printer's Ink was published in 1967) and to be involved in matters of civic and historical concern. From 1966 to 1969, he served on an advisory committee to establish the New Market Battlefield Historical Park and was active with the Virginia Historical Society. Chambers died in 1970.