In early July 1927, the Ohio Department of Highways implemented the system of United States Numbered Highways that had been approved by the states in late 1926. This resulted in the renumbering of many of the state highways to avoid overlaps with the new U.S. Routes and numbers used by both classes of route. In addition to the U.S. Routes, a new State Route 6 was formed, extending West Virginia Route 6 northwest from the Ohio River at Bridgeport to Norwalk. (Route 6 later became U.S. Route 250 in both states.) A number of State Routes - 1, 10, 13, 15, 16, 31, 49, 95, 112, 124, 130, 160, 180, and 223 - were entirely replaced by U.S. Routes or the new State Route 6. The numbers 6, 20, 21, 22, 23, 24, 25, 27, 30, 40, 42, 50, 52, and 127 conflicted with new designations, so the State Routes with those numbers were renamed. Some others - 28, 48, 63, 125, 126, 129, 142, and 263 - were replaced by extensions of other State Routes or new State Routes where not part of a U.S. Route. State Routes 102 and 249 became portions of other routes, but the numbers were reused in the immediate vicinity, 102 for the old alignment of 2 and 249 as part of a three-way route alignment swap in the immediate vicinity. The numbers freed up were reused on other routes.

Later renumberings due to U.S. Routes