Organized in December, 1861, at Calhoun, Ky., under Colonel McHenry, and mustered into the United States service on the 4th day of January, 1862. Taking the field immediately, it was assigned to Cruft's Brigade of Lew Wallace's Division, in which command it fought at the battle of Fort Donelson; loss, 4 killed and 34 wounded. At Shiloh, under command of Colonel McHenry (then in Lauman's Brigade of Hurlbut's Division, it lost 18 killed, 69 wounded, and 1 missing, out of 250 engaged, as officially reported. In April, 1865, the Twenty-fifth Kentucky, having become much reduced in numbers by loss in battle and disease, was discontinued as an organization, and the men were transferred to the Seventeenth Regiment. Soon after this consolidation, Colonel Shackleford and Lieutenant-Colonel Benjamin H. Bristow of the Twenty-fifth were mustered out. The Twenty-fifth, which was in the same brigade, had borne a creditable part in the battles of Fort Donelson and Shiloh, losing at Fort Donelson, 15 killed, 61 wounded, and 12 missing; and at Shiloh, 7 killed and 27 wounded. The rolls of the Twenty-fifth having been turned over with the men, its losses are included in the total loss of the Seventeenth.

At Chickamauga, under command of Colonel Stout, the regiment fought in Beatty's Brigade, Van Cleve's Division, Crittenden's Corps; its loss in that battle was 6 killed, 105 wounded, and 15 missing. Upon the reörganization of the Army of the Cumberland, in October, 1863, the regiment was placed in Beatty's (3d) Brigade, Wood's (3d) Division, Fourth Corps, in which it served throughout the Atlanta campaign. It was mustered out at Louisville, Ky., in January, 1865, the recruits and reënlisted men having been transferred to the Twenty-first Kentucky Infantry. Colonel McHenry was succeeded in December, 1862, by Colonel Stout, who commanded the regiment until its final muster-out.