The regiment was formed as the 127th Ohio Infantry Regiment at Camp Delaware, Ohio. It was re-designated the 5th United States Colored Infantry Regiment, and moved to Norfolk, Virginia, in November 1863, immediately after three months of organization. It served at Norfolk and Portsmouth in the Department of Virginia and North Carolina until January 1864, during which time the unit participated in Brigadier General Edward A. Wild's expedition to South Mills and Camden Court House, North Carolina, from December 5 to December 24 and in action at Sandy Swamp, North Carolina, on December 8.