The largely controlled-access part from Jackson to Starkville connects the state capital with the main campus of Mississippi State University.

The 1933 Road Map of Mississippi shows MS 25 running northward from Macon roughly along the 2019 alignment of U.S. Route 45 to Brooksville, then roughly along the 2019 alignment of U.S. Route 45 Alternate through Artesia and West Point to Muldon, where the 2019 alignment continues in a northeasterly direction.

The last leg to open was the 11.9-mile (19.2 km), $27-million section from the intersection of Highway 19 north of Louisville, Mississippi, to Noxapater Creek in Winston County.

Legally, Mississippi 25 is defined in Mississippi Code Annotated § 65-3-3, as follows: "Begins at or near Jackson, Hinds County, thence in a northeasterly direction to or near Carthage, Louisville and Starkville, thence along U.S. 82 to its intersection with U.S. 45A, thence along U.S. 45A to Muldon, thence to or near Aberdeen, Amory, Smithville, to U.S. 78, thence continuing to Belmont, Dennis, Tishomingo, Iuka and to the Mississippi-Tennessee state line north of Cross Roads, Tishomingo County.

The entire route is in Iuka, Tishomingo County.