The 21st Regiment was organized at Camp Bragg, Oshkosh 5 September 1862 and was sent to Covington, Kentucky for the defense of Cincinnati.

In the battle of Perryville, Kentucky it was mistakenly placed in an exposed position subject to the fire of both friend and foe. As a result of this command blunder it suffered the loss of 179 personnel in killed, wounded and missing.

On 7 December it was ordered to Nashville. With its brigade it repulsed an attack on the supply wagons by 3,500 of Confederate Major General Wheeler's cavalry at Jefferson, the burden of the attack falling on the 21st Regiment. General Rousseau said: "This Regiment, led by its efficient commander behaved like veterans."

It went into the battle of Stone's River the following day, and was sent to the extreme front where they held their position under heavy fire for three days. It encamped at Murfreesboro during the winter and spring.

The Regiment moved south with the Army of the Cumberland in June into Alabama and Georgia and arrived at Chickamauga in time to take part in the second day's fight. An after action battle report stated, "The 21st Regiment never faltered during the whole day, and often the second line would have to face about and drive away the rebels from the rear." On being ordered to retreat it fell back, "Only to the second line of works, where, still fighting and surrounded by the enemy, Lieutenant Colonel Hobart and about 70 officers and men were captured."

The Regiment was placed in reserve at Missionary Ridge and then was stationed on the summit of Lookout Mountain until the spring of 1864. It was in the advance on Resaca in May 1864, and was the last to retire in the evening. At Dallas it remained for six days under fire, its skirmishers being within 50 paces of those of the enemy.

The Regiment next followed Confederate General Johnston in his retreat from Dallas to Kennesaw Mountain, where it charged the enemy's skirmish line and gained position within 150 paces of the main line of Confederate works. It participated in siege, fatigue and guard duty until the fall of Atlanta, took part in the battle of Jonesboro, and then went into camp at Atlanta.

On 1 October it was attached to the 1st Brigade, 1st Division, 14th Army Corps, and pursued General Hood northward. It then rejoined Sherman's Army, took part in the March to the Sea, the siege of Savannah, the Carolina Campaign, and the march to Richmond, Virginia.

It participated in the grand review at Washington, DC where it was mustered out 8 June 1865.