It runs 479 miles (771 km) from Miami in South Florida northwest to the Georgia state line north of the Lake City area.

Within the state, US 41 is paralleled by Interstate 75 (I-75) all the way from Miami to Georgia (on the northern border), and I-75 has largely supplanted US 41 as a major highway.

Like all highways designated by the American Association of State Highway and Transportation Officials (AASHTO) in Florida, US 41 always carries a hidden state road number designated by the Florida Department of Transportation (FDOT): Concurrencies include US 301 between Bradenton and Palmetto, US 92 in Tampa, US 98 and SR 50A in Brooksville, SR 44 in Inverness, US 27 between Williston and High Springs, US 441 between High Springs and Lake City, and US 129 between Hillcoat and Jasper.

The first is US 301 in Sarasota, which runs straight, while US 41 curves to the west towards the John Ringling Causeway, only to turn back north again.

At first, the road runs through Old Seminole Heights where it remains along the border of the Hampton Terrace Historic District until the intersection of Hanna Avenue.

The road leaves Old Seminole Heights by crossing the 1923-built Nebraska Avenue Bridge over the Hillsborough River and enters Sulphur Springs, where one can find such sites as the Springs Theater on the corner of Sitka Street and on the opposite side, the former Tampa Greyhound Track between Bird Street and Waters Avenue.

After this, it curves to the northwest as it begins to run along the west side of the CSX Brooksville Subdivision, and both share an underpass with no access to I-275.

Entering Lutz, the at-grade interchange with the northern end of BUS US 41(Hidden SR 685), is where US 41 becomes six lanes wide.

US 41 only remains in the northwest direction until it encounters the northern end of State Road 597(Dale Mabry Highway) and after turning straight north passing by some naturist resorts, almost instantly intersects SR 54.

Almost a mile (1.6 km) north of that point, the Brooksville Subdivision banks off to the northwest, while US 41 prepares to squeeze between some lakes for which the hamlet is named.

This newly widened section serves as the two gateways to the still under construction Connerton Conner development, and has been slated to become the future end of CR 524(Ridge Road Extension).

Just after that, the gateway to the still under construction Tierra Del Sol development is where the division of the highway ends, although US 41 is still in the process of being widened throughout the northern Tampa Bay suburbs.

While both segments go up and down steep hills in the heart of the city, the Broad Street (eastbound) section runs over an old railroad bridge built in 1936.

North of CR 476, US 41 runs through a portion of the Withlacoochee State Forest where it remains until it crosses the Hernando-Citrus County Line, and gets the name South Florida Avenue.

Meanwhile, the Withlacoochee State Trail continues to run along the east side of the road, often behind the backyards of local businesses and vacant lots.

As the road climbs a slight grade and curves to the left, it approaches a concurrency with SR 44 which also includes a local divided highway named Highland Boulevard in this intersection.

Just before leaving the Inverness city limits, the Withlacoochee State Trail crosses under US 41 and runs along the west side of the road.

On the opposite side of the street, a former Seaboard Air Line Railroad right-of-way follows US 41, although it's no longer noticeable until further north.

The road curves northwest one last time as it prepares to leave Citrus County, passing by some small industrial lots and a local youth athletic park.

After climbing an embankment for a bridge over the Florida Northern Railroad line to the Crystal River 3 Nuclear Power Plant, US 41 intersects County Road 488 then curves northeast as it approaches the Brittan Alexander Bridge over the Withlacoochee River to enter Dunnellon and turns into Williams Street.

The rural surroundings of southern Levy County don't start to diminish until the intersection of Southwest Seventh Avenue.

The US 27/41 concurrency remains only two lanes wide, and it begins to lean to the northwest until briefly shifting straight north before approaching Archer, where it intersects CR 346, then a former Florida Railway and Navigation Company right-of-way, and finally State Road 24.

After SR 24, US 27/41 curves back northwest but head straight northward through State Road 26 in Newberry and both remain as such until US 41 reach High Springs, where US 27 makes a left turn onto State Road 20 towards Perry, Tallahassee, and Havana, while US 41 briefly becomes independent before it joins US 441 towards Lake City and points north.

However, this concurrency only lasts until the intersection of State Road 10A,[citation needed] where SR 47 turns east for a block before joining US 441.

The next block is U.S. Route 90, where the DeSoto Trail heads west, and State Road 100 joins US 41 to the northern extends of Lake City.

Before leaving Lake City's limits, US 41 curves to the northwest to run parallel to a Georgia Southern and Florida Railway line.

Due to the parallel railroad line along the east side, the interchange with Interstate 10 at exit 301 is a pair of quarter-cloverleaf ramps on the southwest and northwest corners of the underpass.

As it leaves the city it returns to the same side of the GSF railroad line it was at earlier, both of which will run through the unincorporated community of Facil where it encounters the southern end of CR 137.

Beyond that, it runs through a strip mining area where the only other community is Camps Still, however after passing through Hillcoat, which is where the northern end of CR 137 can be found, US 41 joins US 129 as well as hidden SR 51.

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