In the U.S. state of Florida, US 23 is concurrent with US 1 south of Alma, Georgia, except in Downtown Jacksonville.

A few miles to the northwest in the Grand Park section of Jacksonville, US 23 runs beneath a railroad bridge originally owned by the St. Johns River Terminal Company (now the Norfolk Southern Springfield Lead) before it meets with US 1/SR 15 (Martin Luther King Jr. Parkway), running concurrently with the highway through the rest of its journey through Florida.

At Callahan, US 1/US 23 meets US 301, beginning a three-way concurrency as the road continues northward through Hilliard and Boulogne toward the St. Marys River, leaving Florida and entering Georgia.

Beginning in 1950, US 23's southern terminus was extended into Florida approximately 319 miles (513 km) south from Atlanta, Georgia, to Jacksonville.

Highways by the Florida Department of Transportation was stopped when the state could no longer use federal funds to replace the signs with anything but the standard black-and-white version.