The regiment was particularly distinguished for its successful attack on Fort Gregg during the 1864 to 1865 Siege of Petersburg, receiving a golden eagle for its flagstaff as a token of appreciation from corps commander John Gibbon.

For much of the first half of 1863, the regiment served at Winchester, Virginia, under Maj. Gen. Robert H. Milroy, and were defeated in their first significant combat action during the Second Battle of Winchester, being pushed off a wooded ridgeline near Kernstown, Virginia, by elements of the Confederate brigade of John B. Gordon on June 13.

The regiment was a part of the Department of West Virginia until December 1864, and it saw action in several fights during the Valley Campaigns of 1864, including the Battle of Opequon or Third Winchester, not far from the scene of its first combat.

The regiment served in the Siege of Petersburg, and on April 2, 1865, distinguished itself for gallantry in a desperate hand-to-hand conflict that resulted in the seizure of Confederate-held Fort Gregg.

"[2] Corporal Andrew O. Apple of Company I was also later awarded the U.S. Medal of Honor for saving the regiment's flag that day.