Prior to their notable actions at Gettysburg in July 1863, the regiment was held in reserve at Antietam in September 1862.

When the regiment came under heavy attack from the Confederate 15th and 47th Alabama regiments (part of the division led by Maj. Gen. John Bell Hood), the 20th Maine ran low on ammunition after one and a half hours of continuous fighting; it responded to the sight of rebel infantry forming again for yet another push up the slope at them by instead suddenly charging downhill with fixed bayonets, surprising and scattering the Confederates, thus ending the attack on the hill and the attempt to flank the hill position and move around the south end of the Federal "fishhook".

Later actions in which the regiment participated included Second Rappahannock Station, Mine Run, The Wilderness, Spotsylvania Court House, the Siege of Petersburg, Peebles's Farm, Lewis's Farm/Quaker Road, White Oak Road, Five Forks, and Appomattox Court House.

[citation needed] The regiment's downhill charge during the Battle of Gettysburg is depicted in the 1974 historical novels The Killer Angels by Michael Shaara (winner of the 1975 Pulitzer Prize in fiction) and Courage on Little Round Top, and was subsequently an important scene in the feature movie made 18 years later, Gettysburg in 1993.

The 2012 music video for the pop song "Some Nights" performed by Fun includes American Civil War Union soldiers serving in the regiment.