North of the intersection with Gibsonton Drive, and over the Alafia River in Riverview, US 301 makes another turn to the northwest, where it crosses I-75 again near Brandon at Exit 254.

Shortly before a diamond interchange with the Selmon Expressway, US 301 intersects with SR 676 at Causeway Boulevard, serving as the route's eastern terminus and providing access to US 41 and US Bus 41.

At Six Mile Creek, an overpass takes US 301 over the tracks of CSX's "A-line", one of the railroad company's main lines through Florida (also used by Amtrak's Silver Star passenger trains), and CR 574, which is only accessible from local connecting roads.

A reconstruction project took place in the early-2000s with provisions for a future ramp from westbound US 92, that are currently being obstructed by the Tampa Bypass Canal and land owned by the Vandenberg Airport.

Near the northern end of Vandenberg Airport, US 301 crosses over a flood channel that runs between the Hillsborough River and Tampa Bypass Canal on the southern border of Temple Terrace.

Former sections of Harney Road are on hills on the northwest and northeast corners, both of which were designed to go around a former railroad bridge for a line that no longer exists.

After passing by a field used for the Tampa Machinery Auctions, US 301 officially runs along the eastern end of Hillsborough River State Park, which it will eventually enter.

Across from the fields of Festival Park, US 301 serves as the eastern terminus of SR 56, and the first major intersection in Pasco County.

This trajectory is short lived though when some local traffic is rerouted along 7th Street, and both roads intersect South Avenue before moving slightly northwest.

From there, US 301 runs along a series of steep hills as a four-lane divided highway with median openings that lack left-turn lanes until it reaches US 98 and the US 98-301 overlap begins.

The first intersection after this is eastern County Road 578 and the next one is at the northern border of the former citrus plant that leads to the Pioneer Florida Museum.

A sure sign the segment is about to end, is when it passed by the Owensboro Junction Trailhead, of the Withlacoochee State Trail.

In Hernando County, US 301 continues its two-lane rural surroundings until it approaches SR 50 in Ridge Manor, which while hardly urban, contains some local businesses, and a large gas station, convenience store and car wash on the northeast corner.

The north end of SR 471 is encountered after this, and then the concurrency with County Road 470 breaks away to head east towards Okahumpka.

Before US 301 enters Wildwood, signs give advanced warnings of the road's approach to the next to last interchange for Florida's Turnpike.

The stretch along the Marion-Sumter county line serves as the unofficial western boundary of The Villages, the large Central Florida retirement community.

Near the right of way for the formerly proposed Cross Florida Barge Canal, the median for the road widens, but narrows back down to normal again.

The highway reaches the heart of Ocala at the intersection with SR 40 (West Silver Springs Boulevard).

Five blocks later, US 27 leaves the US 301-441 overlap and takes SR 500 with it as they head northwest towards Williston, Perry, Tallahassee, and points north.

After US 301 crosses the Alachua County Line it runs along a four-lane causeway through marshland before entering Island Grove, where the road intersects with another former section (Southeast 201st Terrace) and CR 325, which leads to Marjorie Kinnan Rawlings Historic State Park.

Intersections with CR 1474 in Campville and SR 26 in Orange Heights are at-grade interchanges, and the railroad line breaks away from the road moving straight north.

US 301 would head back to the northwest in Shenks, where it would otherwise take drivers straight into Lake Altho and those tracks can be found again, beneath the interchange with SR 24 in Waldo.

Since US 301 is a popular short cut between Northeastern Florida and the Gulf Coast region, a number of towns along the road have been notorious speed traps.

Many have accused the police in Waldo, Starke, Lawtey, and others of giving tickets simply to raise money.

After passing through Fiftone the road encounters a large railroad yard on the east side before the partial-cloverleaf interchange with I-10, before entering Baldwin.

In September 2020, the Florida Department of Transportation opened the Baldwin Bypass around the western edge of town.

US 301 remains a two-lane rural highway until it reaches the northern end of the Baldwin Bypass, and then turns right to continue north.

Here, SR A1A/200 continue running along East First Avenue toward Fernandina Beach, while US 301 becomes part of a three route overlap with US 1-23 between Callahan, and Folkston, Georgia.

Routes by the Florida Department of Transportation was stopped when the state could no longer use federal funds to replace signs that were not in compliance with the Manual on Uniform Traffic Control Devices (MUTCD) per 23 CFR 655.603.