SR 121 is one of the major routes of a three-state highway 121 that totals approximately 475 miles (764 km) from Lebanon Station, Florida, to Rock Hill, South Carolina.

The road makes a sharp left curve to the northeast where it travels through desolate farmland; the only resemblance to a major intersection is CR 326.

Within the town of LaCrosse, SR 121 shares a short concurrency with SR 235, then has a level crossing with the CSX Brooker Subdivision, a former Seaboard Coast Line railroad line that leads to the Crystal River 3 Nuclear Power Plant.

The road also runs parallel to another rail-trail along the former Jacksonville and Southwestern right-of-way in Raiford, then serves as the western terminus of SR 16 before leaving that community.

FDOT widened the road within the city, which included the creation of a retaining wall along the University of Florida Golf Course that has since become a target for graffiti artists.

SR 331 terminates at the east end of the overlap of SRs 20, 24, and 26, which also serves as the terminus of Florida Truck Routes 24 and 26.

At this point, the truck routes turn west overlapping SR 222 and intersection Florida State Road 20.