Shackelford was appointed colonel of the 25th Kentucky Infantry on January 1, 1862. His regiment joined Lew Wallace's division during the Battle of Fort Donelson. Exposure to the elements at Fort Donelson forced Shackelford to resign on March 24, 1862, on account of poor health. During his convalescence, Shackelford helped recruit another regiment of Kentucky volunteers which was mustered in as the 8th Kentucky Cavalry with Shackelford as colonel. A few months later, on January 1, 1863, he was promoted to Brigadier General of Volunteers and assigned to command the 1st Brigade, 2nd Division, XXIII Corps.