[2] The Regiment fought at the Battle of Antietam[1][3] under the leadership of Colonel Jacob C. Higgins[4] less than six weeks after being recruited in Blair, Cambria and Huntingdon Counties.

Certain that the five new regiments of Williams' First Division would run away if deployed in line of battle, Mansfield ordered the First Brigade to lead in a tight formation known as close column of companies.

Three members of the 125th Pennsylvania (Coho, Edmundson, & Rudy, along with two others) helped him from his horse, carried him to the rear on a chair formed from muskets, and turned him over to a surgeon.

After a delay for re-organization, the 125th Pennsylvania moved forward to support Monroe's First Rhode Island Battery (I Corps, 1 Div) diagonally across the intersection of Smoketown Road and Hagerstown Pike at about 8:45.

"[23] In his memoirs General William T. Sherman decried a systematic problem which the 125th Pennsylvania fell prey to at Antietam, along with many other Regiments in their initial battles: Significantly, Antietam was the last battle fought in the east without the construction of field fortifications, although naturally occurring features, such as the Sunken Road and the quarry holes above the Burnside Bridge, were exploited as rifle pits.

The 125th Pennsylvania Volunteer Infantry Monument on the Antietam Battlefield was dedicated on September 17, 1904, and is located on Confederate Avenue behind (West) of the Dunker Church.

[27] During the 125th Pennsylvania's withdrawal from the indicated position in the West Woods, one of the most dramatic events in the regiment's history began with the regimental color-bearer, Sergeant George Simpson who stands immortalized in granite on top of the monument: By October 30, 1862, the 125th Pennsylvania had been reassigned to the 2nd Brigade, 1st Division (XII Corps)[29] under Brigadier General Thomas L. Kane and newly positioned at Loudon Heights, VA, near Harper's Ferry, for extended drilling.

Meade's (V) 1st & 3rd Divisions were advancing unimpeded toward a strategic imperative, occupation of Bank's Ford on the Rappahannock River; nonetheless, Hooker called a halt to all operations and ordered a retreat into defensive positions around Chancellorsville.

Private Hicks noted, "From this force we could hear an occasional command, 'Close up,' 'Steady, men,' and like words, and now and then we could catch glimpses of the gray-clothed ranks moving with steady steps, with arms at right shoulder and paying no attention either to us or to our skirmish line in front".

While Virginians led by General William Mahone attacked the 2nd Division of the XII Corps, Confederate artillery on the Orange Plank Road and at Hazel Grove sent shells screeching into Slocum's line from the rear.

[34] Constructing effective breastworks near the Chancellor Mansion and Tavern, the 125th Pennsylvania's Regimental losses were limited (5 killed, 12 wounded, and 10 captured).

[35] The fiercest fighting of the Chancellorsville Campaign occurred on May 3, including action at Salem Church and Fredericksburg, and produced the second bloodiest day of the Civil War.

Shortly after the Regiment mustered out and returned home, the Confederate victory at the Second Battle of Winchester on June 13–15, 1863, opened the doorway for the invasion of Pennsylvania.