U.S. Route 321 is a spur of U.S. Route 21. It runs for 526 miles (847 km) from South Carolina to Tennessee. The northern terminus of U.S. 321 is between Lenoir City and Oak Ridge, Tennessee, at Interstate 40 exit 364, which is about 4 miles (6.4 km) west of Interstate 40's junction with Interstate 75. The southern terminus is in Hardeeville, South Carolina at the intersection with U.S. Route 17, less than a mile away from Interstate 95 and 15 miles (24 km) north of Savannah, Georgia.

Because of its unusual "north–south–north" route, U.S. 321 intersects both Interstate 40 and U.S. Route 70 three separate times.

U.S. 321 provides direct access between Savannah and Columbia, serving as an alternate to Interstate 95 and Interstate 26. Starting in Hardeeville, U.S. 321 as a mostly 2 lane highway goes through sparsely populated areas and small towns including Estill, Fairfax, and Denmark, heading in a rather straight and northward direction into the Columbia area, widening to a 5 lane highway right after Neeses, then narrowing down to a 2 lane highway right after the town of North and widening to a 5 lane highway again after Swansea and staying that way until it merges with its parent route U.S. 21 in Dixiana. In Columbia, the route stays concurrent with U.S. 21 through Cayce, over the Congaree River on the Blossom Street Bridge, turning left onto Huger Street, right onto Elmwood Avenue, and left onto North Main Street. The roads separate at Hyatt Park, with U.S. 321 staying west of Interstate 77 and U.S. 21 north of Columbia. The route goes through to communities of Winnsboro, Chester, York, and Clover before entering North Carolina at Bowling Green.

U.S. 321 is an important route in Western North Carolina. It provides an alternative north–south route to Interstate 77, which passes through a busy metropolitan area, and US 221, which is more twisty drive up the Appalachians. Travelers from South Carolina who are going to points west of Charlotte and want to avoid the traffic of I-77 may choose to enter the state on U.S. 321.

The highway traverses through seven counties: Gaston, Lincoln, Catawba, Burke, Caldwell, Watauga, and Avery. U.S. 321 has six control cities: Gastonia, Lincolnton, Hickory, Lenoir, Blowing Rock, and Boone.

U.S. 321 is a multilane highway beginning at the state line. After 7 miles (11 km), it enters the Gastonia area. Traffic on northbound 321 through Gastonia is generally not as heavy as traffic on southbound 321 north of Interstate 85, where it may back up for a couple of miles as drivers wait to turn onto I-85.

The road becomes a freeway at C. Grier Beam Boulevard in Gastonia, just south of NC 275/NC 279. It remains a freeway until just north of its junction with US 70 in Hickory. The freeway bypasses a number of cities that mainline U.S. 321 once passed through. Today, U.S. 321 Business follows the original 38 miles (61 km) route, that serves Maiden, Newton, and Conover; but the freeway is a more direct route to the mountains, and the business route is generally used by local traffic only.

Traffic is usually heavy between Hickory and Lenoir. The North Carolina Department of Transportation (NCDOT) has a long-term plan to widen the road to six lanes, which is estimated to be completed by 2015. However, the project will probably take considerally longer. The first stage of the project (NCDOT project B-4450), replacing the bridge over the Catawba River, was previously estimated to be completed by 2011. However, the NCDOT project page shows that right-of-way acquisition will not begin until 2012.

North of Lenoir, U.S. 321 is a four-lane divided highway. Currently the highway becomes a two-lane road again in northern Caldwell County. NCDOT project R-2237 will widen the road the four lanes through Blowing Rock. The widening of U.S. 321 through Blowing Rock has been a controversial project. Many residents of the town feel that a four-lane highway will destroy the character of the small town, and they proposed several bypass alternatives. NCDOT has selected the widening as its preferred alternative, but added several features such as underground utilities, sidewalks, landscaping, and rock walls to make the widening more palatable to the residents who are opposed to it. These features are not present on the two-lane highway that is being replaced.

North of Blowing Rock, U.S. 321 is already a four-lane highway up to King Street in Boone; thus, R-2237 will make 321 a four-lane highway from the South Carolina state line to downtown Boone. Currently, U.S. 321 meets US 421 at King Street and overlaps it over King Street to leave town to the west. NCDOT project R-2615 is proposed to widen the U.S. 321/421 concurrency from this junction to their junction near Vilas, although this project is currently unfunded. There is a feasibility study (FS-0511A) in progress to examine the possibility of widening U.S. 321 from the junction near Vilas to the Tennessee state line. If both of these projects were completed, then U.S. 321 would be a four-lane highway through the entire state.

U.S. 321 "changes direction" in Elizabethton: the road is designated as southbound in both directions driving away from its intersection with US 19E. It is conterminous with US 11E through portions of Washington and Greene counties. In Greeneville, the highway continues south into Cocke County, crossing I-40 at Newport. The stretch of US 321 from Newport south to Cosby and southwest through Pittman Center to Gatlinburg parallels the northern boundary of the Great Smoky Mountains National Park and is quite scenic. From a point between Newport and Cosby to the intersection with the Little River Road, it is concurrent with TN 73. In Pigeon Forge, concurrent with U.S. Route 441/Great Smoky Mountains Parkway, US 321 is a divided, multi-lane traffic-laden thoroughfare where it serves as the city's main route, lined with hotels, outlet malls and amusement facilities. Veering southwest of Pigeon Forge, the highway takes on a more rural character, and narrows down to 2 lanes as it traverses Wears Valley until its junction with Tennessee State Route 73/337 (the Little River Road) at the Townsend entrance to the Great Smoky Mountains National Park. It is concurrent with Tennessee 73 from this junction to its terminus. With the exception of a 3-mile (4.8 km) section between Townsend and Maryville, the remaining portions of the highway through Blount County are a minimum of 4 lanes and usually a divided highway until it reaches Fort Loudon Dam in Loudon County where it crosses the Tennessee River at just south of Lenoir City, and has a wrong-way concurrency with Tennessee State Route 95 from a point several miles south of Lenoir City to its terminus at I-40.

Current plans call for a new bridge crossing over Fort Loudoun Lake and Fort Loudoun Dam along with widening of 1.4 miles (2.3 km) of related highway, with construction underway.[citation needed] Though sections of the highway are already four-laned, remaining portions through Loudon County will also be widened as part of the project, meaning the highway would be a four-lane semi-divided thoroughfare from Interstate 40 and Interstate 75 to the Great Smoky Mountains National Park, except for the 3-mile (4.8 km) stretch near Walland, TN.

In addition to the widening project, there have also been talks of a freeway-to-freeway connection with Interstate 85 in Gastonia that would greatly relieve traffic flowing through the junction. Currently, U.S. 321 is a surface road while passing through the interchange, but shortly becomes a freeway north of there. However, in order to extend the freeway to I-85, the few remaining at-grade intersections and traffic lights would have to be eliminated. An interchange is planned for the proposed Garden Parkway, which would terminate at the freeway section of U.S. 321 near Dallas.

Currently, there are three U.S. Route 321 Business loops in South Carolina:

The 4.2 miles (6.8 km) route that goes through the downtowns of Winnsboro Mills and Winnsboro, also known as Columbia Road and Congress Street.

The 3.5 miles (5.6 km) route that goes through downtown Chester, also known as Columbia Street and Center Street.

The 2.5 miles (4.0 km) route that goes through downtown York, also known as Congress Street and Kings Mountain Street.

Currently, there is one U.S. Route 321 alternate route active and one decommissioned:

U.S. Route 321A is an alternate route of US 321 in western Caldwell County. It services the towns of Granite Falls, Sawmills, and Hudson before reconnecting with the main US 321 in Lenoir.

Established between 1954–58, it followed the old mainline US 321 route through downtown Blowing Rock, via Main Street. It was renumbered to US 321 Business in 1960.

Currently, there are two U.S. Route 321 Business loops active and one decommissioned:

The 38 miles (61 km) route connects the towns and cities of High Shoals, Lincolnton, Maiden, Newton, and Conover. The route was the original US 321 before the freeway was completed in the mid-1990s; it was first signed as NC 155, but was later re-branded US 321 Business because of opposition from Newton and Conover of the route being downgraded to a North Carolina highway.

The 2 miles (3.2 km) was originally signed between 1954–58 as US 321A, it was re-branded in 1960 as business route.