James S. Negley was born in East Liberty in Allegheny County, Pennsylvania. He was educated in public schools and graduated from the Western University of Pennsylvania. He served in a Pennsylvania volunteer regiment during the Mexican-American War. After the war, he became a farmer and horticulturist.

At the outbreak of the Civil War, Negley raised a brigade of Pennsylvania volunteers and was appointed its brigadier general, serving under Robert Patterson in the Shenandoah Valley in 1861. In October, he was placed in command of a division in the Deptartment of the Ohio. He commanded the Union forces at the Battle of Chattanooga during the Confederate Heartland Offensive. In November of 1862, he was appointed major general of volunteers, and his division became the 2nd Division, Center Corps at the Battle of Stones River. On the second day of fighting, he led a successful counterattack against John C. Breckinridge on the Union left flank. He commanded his division during the Tullahoma Campaign and the Battle of Chickamauga. After the defeat at Chickamauga, Negley was relieved of command, but was acquitted of any wrongdoing during the battle. After serving on several administrative boards, he resigned in January of 1865.

After the war, Negley was elected as a Republican to the United States Congress in 1869. He served on the board of managers of the National Home for Disabled Volunteer Soldiers from 1874-1878. He was re-elected to Congress and served until 1887. After retiring from politics, he was engaged in railroading. Negley died in Plainfield, New Jersey.