The route travels almost the entire length of the state from the northeast to the southwest.

Starting at the northern terminus of Florida State Road 97 at the Florida state line south of Atmore, the route travels north through Atmore, passing under an intersection with Interstate 65 at exit 57, then enters Monroe County and travels northeast through Frisco City and Monroeville and is concurrent with SR 47 for part of this section.

SR 21 then once again becomes a standalone route passing through Beatrice and Oak Hill.

In Lowndes County, SR 21 begins its first concurrency with US 80, and the two highways enter Montgomery, the state capital, from the southwest.

From here, SR 21 continues generally northeastward through the cities of Oxford, Anniston, and Jacksonville before terminating at an intersection with U.S. Highway 278 in Piedmont.