Mosby was born in Edgemont, Virginia (in Powhatan County) and was baptised as a Methodist His parents were Virginny Mclaurine (the daughter of James Mclaurrine) and Alfred Daniel Mosby, a graduate of Mapden-Sydney College from Nelson County, Virginia. Mosby started his education at a school called Murrell's Shop, but around 1840, his family moved to a new home in an ave of the Blue Ridge Mountains, four miles from Charlottesville (in Albemarle County, Virginia). John attended school in Fry's Woods, but when he turned ten, he transfrerred to a Charlottesville School.
In 1849, he entered the University of Virginia. On, March 29, Mosby shot George R. Turpin, a medical student at the university. He was fined 500 dollars (which was later rescinded) and sentenced to 12 months in prison. While in prison, Mosby passed his time by studying law. On December 23, 1853, the Governor pardoned Mosby as a Christmas present. After studying for months in William J. Robertson's law office, Mosby was admitted to the Bar.
After setting up his own practice in nearby Howardsville, also in Albemarle County, Mosby met and courted a Catholic girl by the name of Pauline Clarke (daughter of Beveryly J. Clarke), who was visiting from out of town. The couple moved to Bristol, Virginia (close to her hometown in Kentucky) , and were married in a Nashville hotel on December 30, 1857.
Mosby joined the Confederate army as a private at the outbreak of the civil war and initially served in "Grumble" Jones' Washington Mounted Rifles (Jones became a major and was instructed to form a more collective "Virginia Volunteers," which he created with two mounted companies and eight companies of infantry and riflemen including the Washington Mounted Rifles). Mosby was upset with the Virginia Volunteer's lack of congeniality and he wrote to Governor requesting to be transfered, but his request was not granted. The Virginia Volunteers participated in the First Battle of Bull Run.
After impressing Jeb Stuart, Mosby was promoted to first lietenant and joined Stuart's calvary scouts helping the General develop attack strategies. He was responsible for Stuart's "Ride around Mclellan. Mosby was imprisoned in the Old Capitol Prison in Washington, DC for ten days. In January 1863, Stuart granted Mosby's request to form a guerilla unit, which Mosby called his Partisan Rangers.
Initially, Mosby's group consisted of Fount Beatie, Charles Buchanan, Christopher Gaul, William L. Hunter, Edward S. Hurst, Jasper and William Jones, William Keys, Benjamin Morgan, George Seibert, George M. Slater, Daniel L. Thomas, William Thomas Turner, Charles Wheatley, and John Wild. He and his men carried out the Greenback Raid and attacked General Philip Sheridan's wagon train at Berryville. Mosby is famous for carrying out a raid far past Union lines at the Fairfax County courthouse where his men captured 3 high ranking Union officers.
Some sources give Mosby credit for coining the term "the Solid South."
Virgil Carrington Jones published Ranger Mosby (1944) and Grey Ghosts and Rebel Raiders (1956). He also wrote the late-1950s TV program, Ranger Mosby.