This Regiment was organized at Camp Franklin, near Dubuque, and mustered in from 18 to 23 August 1863. It left the state 19 September for St. Louis and reached Rolla on the 23rd. At Salem it was attached to a Brigade consisting of the 33rd Missouri, 99th Illinois, and some artillery and Cavalry, under command of General Fitz-­Henry Warren.  The Brigade train was surprised and captured on the night of 24 November when 15 of the Regiment, forming a part of the guard, were killed or captured. The enemy escaped without damage after burning the train, the Regiment being at Hartville, 16 miles away.

A detachment of the Regiment formed a part of the force sent to Springfield under Colonel Merrill in January 1863, to reinforce General Brown, who was threatened with attack, and in fact sustained one before the messenger had reached Hartville. The detachment returned to Hartville on the 11th and found a large body of the enemy at that place.

The less than 1,000 men under Merrill gave battle at once, fought from noon until night, repulsed every charge and inflicted a loss of 300 in killed and wounded. The detachment of the 21st did not receive the order to retire at the close of the day and fought until long after dark, sustaining alone three charges, which were repelled. This was the first time the Regiment had been under fire and it received high praise for its coolness and excellent work.  Two hundred and fifty of the Regiment left at Houston formed a part of a force that marched to Hartville on the 12th on learning of the engagement. It moved the 30 miles intervening, and returned to Houston, having made the distance of 60 miles in a little over 24 hours. The remainder of the command reached there on the 17th.

At West Plains the Regiment was assigned to the 2nd Brigade, 2nd Division, Army of Southeast Missouri. Without protection, half starved and nearly frozen, the Army concentrated at that point and remained until 8 February by which time the commanding officer found it necessary to move in order to preserve his men. The command marched to Eminence thence to Iron Mountain, many of the men being without shoes, their feet wrapped in pieces of ragged clothing, with hardly a mouthful of food. From Iron Mountain it moved to St. Genevieve, which was reached on 11 March. One wing of the Regiment under Major Van Anda embarked on the 26th, the other on 1 April for Milliken's bend, where it was assigned to the 2nd Brigade, 14th Division, 13th Army Corps, commanded by Brigadier General John Alexander McClernand.

It took part in the battle of Port Gibson where the Division commander referred to it as, being "first in battle and one of the last to leave the field." Its losses were light. It was in reserve at the battle of Champion's Hill, at the Big Black River it accompanied the 23rd Iowa on a terrible charge across the river bottoms and the bayou, over the enemy's works, and captured over 1,700 prisoners and 18 guns. Colonel Merrill was severely wounded and the Regiment lost 13 killed and 70 wounded out of less than 300 engaged. 

At Vicksburg it took part in the siege; and in the assault of 22 May it lost 113 in killed and wounded out of 280 who took part. Lieutenant Colonel Dunlap, who had been wounded at Port Gibson was on the field and was killed after the assault, and Major Van Anda was wounded during the charge. At the siege conclusion the Regiment took part in the siege of Jackson and in the destruction of the railways and public property at that place.

On 13 August it moved to Carrollton, Louisiana, then to Brashear City, Berwick City, then up Bayou Teche to Vermillion Bayou, where it remained for a month, guarding a bridge and performing heavy picket duty. In November it moved to New Iberia, Berwick City and Brashear, reached Algiers on the 21st and embarked the following day for St. Joseph's Island, Texas. On 13 March the Regiment moved to Matagorda Island, where it remained until June. On 26 July it moved to Morganza, on 3 September it went to the mouth of the White River, then to St. Charles, Devall's Bluff and Memphis. There it was assigned to the 1st Brigade, Reserve Corps Division of West Mississippi. 

The Regiment then took part in a march into the interior of Tennessee in December, embarked for Kennerville on 1 January 1865, and on 5 February, sailed for Dauphin Island, Alabama. From there it proceeded to Fort Morgan, then moved to Mobile Bay and took part in the siege of Spanish Fort and Fort Blakely.

It was mustered out at Mobile, Alabama in July 1865.