The Third Infantry was organized at Fond du Lac, WI and mustered into the service of the United States on the 29th day of June, 1861. The regiment left the state on the 12th day of July following, reporting at Hagerstown, MD, from which point it marched to Harper's Ferry. Shortly thereafter the Third was assigned to Patterson's Army and its first year of service was principally upon the upper Potomac and in the Shenandoah Valley, participating in a number of engagements, the most important of which were Winchester and Cedar Mountain.

When the Union army under General Pope was being pressed northward by the Confederates in August 1862, the portion of the army in which the Third was serving was united with the army under General Pope, and shortly thereafter General McClellan assigned as Commandeer in Chief, and as a part of this force the Third participated in the battle of Antietam. In the early part of 1863, with the Army of the Potomac, participated in the campaign of Chancellorsville, VA, and the subsequent battle of Gettysburg, July 1-3, 1863. On July 13, 1863, the Third was sent to the city of New York to assist in quelling the draft riots in that city, and encamped in the City Hall park until September 5, when the regiment returned to Virginia. In the latter part of that month the Twelfth Corps, of which the Third was a part, was transferred to southern middle Tennessee as a part of the Army of the Cumberland. The Third was stationed at Stevenson, AL, from the 3rd day of October, and during the latter part of that year and the early part of 1864 was engaged in guarding important lines of railroad communications in southern middle Tennessee and northern Alabama.

On the reorganization of the Army about Chattanooga for the advance upon Atlanta under General Sherman, the Third was assigned to the Second Brigade of the First Division of the Twentieth Army Corps commanded by General Joseph Hooker, and as a part of this organization participated in the Atlanta Campaign from Chattanooga to Atlanta, May 1 to September 8, 1864; the "March to the Sea", Atlanta to Savannah, November 15 to December 21, 1864, and from Savannah through the Carolinas, January 1 to April 26, 1865, until the surrender of the Confederate forces under General Johnston April 26, 1865. Thereafter the Third participated in the Grand Review at Washington. On June 15 was transferred to Louisville, KY, was mustered out of the service of the United States on the 18th of July, 1865, returned to Madison, WI on the 23rd of July and was shortly thereafter disbanded.