Raised mostly in northern and central Pennsylvania in mid-1864, the regiment initially served with the Army of the James during the Siege of Petersburg, holding trenches at Bermuda Hundred.

In late November it transferred to the Army of the Potomac and during the northern hemisphere spring campaign fought in the counterattack during the Battle of Fort Stedman and the Union breakthrough at Petersburg.

During last days of the Appomattox Campaign it guarded the supply line, and participated in the Grand Review of the Armies following the end of the war before mustering out.

The ten companies of the regiment moved to Camp Curtin near Harrisburg, where they were organized on 8 September under the command of volunteer officer Colonel Robert C. Cox, the former major of the 171st Pennsylvania Infantry.

[2] After completing its organization, the 207th Pennsylvania was sent to the front on 12 September and spent the next four days waiting for orders before it was assigned to the Provisional Brigade of the Defenses of Bermuda Hundred of the Army of the James in the Siege of Petersburg.

[2] Fraternization between the opposing pickets was routine, but this state of affairs ended when Confederate division commander George Pickett launched a night attack on 17 November.

Shortly after 03:00, it advanced from the camp and formed in a line of battle directly in front of Fort Sedgwick with its left on the Jerusalem Plank Road, the forward regiment of the brigade.

[5] The regiment repulsed multiple counterattacks in fierce fighting,[5] during which Cox temporarily relieved the ill Mathews in command of the brigade at 10:00.

[4] In 1866, Cox was recognized "for gallant and meritorious services at Forts Stedman and Sedgwick, Va., April 2, 1865" when he was nominated by President Andrew Johnson and approved by the U.S. Senate for the rank of brigadier-general by brevet.