Shackleford enlisted in January 1862 to receive his first commission as a colonel in the 25th Kentucky Infantry. He served at Fort Donelson, but he resigned in March due to ill health. By September he returned to service as a colonel of the 8th Kentucky Cavalry. In the following year, he was promoted to brigadier general during the pursuit of Confederate General John H. Morgan and his cavalry. In September 1863, Shackleford was wounded in the foot; however, he remained on the field in command of the cavalry in the defense of Knoxville. He resigned in January 1864.