Chambers was born at Bromley Bridge, Somerset County, New Jersey, on October 6, 1780 to Roland Chambers (1744–1821).
He attended the public schools and the Transylvania Seminary at Lexington, Kentucky. In 1794 he moved with his father to Washington, Mason County, Kentucky. After studying law he was admitted to the bar in 1800 and commenced practice in Washington, Kentucky. Chambers served as aide-de-camp to General William Henry Harrison in the War of 1812 and was at the Battle of the Thames. He served as a member of the State house of representatives in 1812, 1815, 1830, and 1831. In 1825, Chambers was appointed judge of the Kentucky Court of Appeals. He resigned in 1827.
He was elected as a pro-Adams candidate to the Twentieth Congress to fill the vacancy caused by the resignation of Thomas Metcalfe and served from December 1, 1828, to March 3, 1829; elected as an Anti-Jacksonian to the Twenty-fourth Congress, and reelected as a Whig to the Twenty-fifth Congress (March 4, 1835 – March 3, 1839); chairman, Committee on Claims (Twenty-fifth Congress).
Chambers represented the counties of Pendleton, Bracken, Robertson, Nicholas and Bourbon.
Chambers was appointed Governor of the Iowa Territory in 1841, serving until 1845. He was then commissioner to negotiate a treaty with the Sioux Indians in 1849. He died near Paris, Bourbon County, Kentucky, September 21, 1852 and was interred in the family burial ground at Washington, in Mason County, Kentucky.
He married Margaret Taylor (b. May 22, 1781), daughter of Major Ignatius Taylor (1742–1807), on June 16, 1803. She died on March 4, 1807. They had no surviving children.
He married secondly, on October 29, 1807, to Hannah Lee Taylor (January 9, 1791 – November 11, 1832), daughter of Major Ignatius Taylor with his second wife, Barbara Bowie (1756–1805). Hannah was a half sister to John's first wife Margaret. John and Hannah had twelve children; Margaret Taylor (1808–1863), Joseph Sprigg Taylor, Hannah Lee Taylor, James Taylor, Matilda Taylor, Francis Taylor, Jane Taylor, Mary Taylor, Laura Taylor, John Taylor, James Taylor, Henry Taylor, Lucretia Taylor.
Chambers died September 21, 1852 at the age of 71 in Paris, Kentucky and is buried in Chambers Family Cemetery in Old Washington, Mason County, Kentucky.