This Regiment was organized at Camp Randall in June and July 1862, and was mustered into US service 23 August. It left the state on 30 August, going first to Benton barracks, St. Louis, then to Rolla, Springfield and Cassville, Missouri, and Cross Hollow, Arkansas, the enemy falling back at its approach.

On 4 November the Regiment started for Wilson's Creek, Arkansas where it joined General Totten's command at Ozark on the 11th and reached Wilson's Creek on the 22nd.

In December it made a forced march of 100 miles in three days to Fayetteville; was in the battle of Prairie Grove where it charged the heights through underbrush and captured a battery of 6 guns. Due to the deadly crossfire of five Regiments of the enemy, the 20th was forced to retire with a loss of 209 in killed, wounded and missing. General Herron wrote Governor Salomon, "I congratulate you and the state on the glorious conduct of the 20th Wis. infantry in the great battle of Prairie Grove."

The Regiment wintered in Missouri, then traveled to Vicksburg on 3 June 1863 where took position in the trenches, remaining there until the city's surrender. Afterward the Regiment occupied Yazoo City, Mississippi as provost guard.

In Louisiana, it arrived in Port Hudson, 21 July and remained until 13 August when it was ordered to Carrollton and Morganza. During the march to the Atchafalaya River, the Regiment was ambushed in Louisiana on the Simsport Road but lost only one man.

After traveling to the Rio Grande on the steamer "Thomas A. Scott", it was employed in fatigue, garrison and picket duty at Fort Brown, Texas and was sent to Matamoras, Mexico 12 January 1864, to protect the American consul and remove American goods.

On 28 July the Regiment left for New Orleans, took up position at Navy Cove on 11 August, and assisted in capturing Fort Morgan, Louisiana.  

On 14 December it sailed for Pascagoula, Mississippi. Subsequently, while traveling under orders to Mobile, Alabama the Regiment routed a body of the enemy at Franklin Creek, and was later engaged in the vicinity of Mobile during the winter.

Its next significant action was 27 March against Spanish Fort where was subjected to enemy fire several days and nights. After the surrender of Mobile it remained in the area until June.

It was then ordered to Galveston, Texas where it was mustered out 14 July.

Its original strength was 990. Gained by recruits, 138; substitute, 1; total, 1,129. Loss by death, 227; desertion, 41; transfer, 115; discharge, 222; mustered out, 524.