However the beginning of its run is a short curve towards the north with a connecting County Road 55A leading to southbound US 19/98/Alt-27, and only begins to move it its signed cardinal direction in Wilcox east of the junction between three segments of the Nature Coast State Trail.

After running through the historic district, it intersects with US 27-41 (unsigned SR 45), and continues to maintain the same street name even as it passes through the communities of Jonesville and Tioga before encountering the interchange with Interstate 75 just west of Gainesville.

Leaving the preserve area and passing through farmland, the road later crosses US 301 in Orange Heights, although a realignment of SR 26 south of the existing intersection was built by the Florida Department of Transportation between 2010 and 2011 which includes an interchange.

It is locally known as West Newberry Road and Southwest Second Avenue, and runs along the northwest corner of the University of Florida.

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.