It runs 88 miles (142 km) from Chiefland north to the Georgia State Line in Levy, Gilchrist, Suwannee, and Hamilton Counties.

The road is concurrent with hidden SR 49, running northeast until it reaches Northwest 30th Avenue and turns straight north where it remains through the rest of Levy County.

From the Santa Fe River Bridge, the first intersection is 296th Street, and all subsequent intersections are equally minor until it reaches Hildreth, where it crosses the Suwannee River Greenway Trail,[3] then encounters the east end of a concurrency with US 27 (hidden SR 20) which is also the northern terminus of SR 49 and the southern terminus of CR 49.

A slight curve to the southwest is where both routes officially enter Branford, Florida where US 129 turns north onto Hidden SR 249, while US 27 continues west across the Frank R. Norris Bridge over the Suwannee River into Grady towards Mayo, Perry, Tallahassee, and beyond.

Within the historic Live Oak the road is named Ohio Avenue, and continues to run in a straight south-to-north line.

Before leaving the city limits, the road finally becomes four lanes wide and eventually gains a divider as commercial development begins to dot the landscape, serving as an indicator that it is designed to accommodate motorists from the nearby Interstate 10, and sure enough, the road finally has a diamond interchange with I-10 at exit 283.

The only intersection of note in this area is with the western terminus of CR 136A, a suffixed alternate of SR 136, which is also named "County Road 136 Scenic."

Unlike in Suwannee County, only a hint of the right-of-way of the former segment of US 129 can be found before the road turns straight north before letting go of its overlap with CR 132, which runs east.

US 129 then approaches a Love's truck stop and a pair of quarter-cloverleaf ramps on the southeast and northwest corners above its interchange with I-75 at exit 451.

Resuming its northward agenda, which US 41 will do further on, US 129/SR 100 runs through the forests of northern Florida interrupted by the occasional private house and farmland.

The only real intersection of any significance it encounters is CR 148 in Rawls, where it also crosses the same Georgia Southern and Florida Railway line that followed US 41 from Lake City.

Eleven years later, the segment south of Branford would be rerouted in the proper direction along US 27 and make a right turn along SR 49 toward Trenton and Chiefland, while the old US 129 would become US 129 Alternate (see below).