U.S. Route 421 is a spur route of U.S. 21. It runs for 941 miles (1,514 km) from Michigan City, Indiana, at U.S. 20, to Fort Fisher in North Carolina. The highway goes through the cities of Indianapolis, Indiana, Lexington, Kentucky, Boone, North Carolina, Winston-Salem, North Carolina, Greensboro, North Carolina and ends in Wilmington, North Carolina.

U.S. Route 421 begins at Fort Fisher, North Carolina, and heads generally to the northwest to Michigan City, Indiana. Along the way, it passes through Bristol, Tennessee, and Virginia, Lexington, Kentucky, and Indianapolis, Indiana.

US 421 begins at Fort Fisher and heads through North Carolina's southeastern beaches to Wilmington. It then heads in a northwest direction through Clinton, Dunn, Lillington, Sanford and Siler City to the Piedmont Triad region. Major highway junctures between north of Wilmington and Greensboro are NC 11, I-95, U.S. Route 401, US 1, US 64 and I-85. In Greensboro, it follows the Greensboro Urban Loop paired with Interstate 85 south of Greensboro and I-73 northwest of Greensboro. It then connects to I-40 towards Forsyth County as it becomes its own freeway along the former Interstate 40 alignment known as Business 40. Major intersections are NC 66, US 158 and US 52 between Greensboro and Winston-Salem. After Winston-Salem, it intersects I-40 once again but continues westerly passing through Lewisville, Yadkinville, Wilkesboro and Boone. Major highway junctions between Winston-Salem and Boone are US 601, I-77, NC 16, Blue Ridge Parkway, US 221 and US 321. US 421 continues westerly into Tennessee towards Mountain City, Tennessee. US 421 is a limited access freeway through Siler City northward. US 421 is a divided highway from north of NC 49 to the Greensboro Urban Loop. US 421 is a limited access freeway from this point all of the way to Wilkesboro.

US Highway 421 enters Tennessee from North Carolina in Trade. It is the oldest crossroads community in Tennessee and is the easternmost part of the state. The road "as the crow would fly" is a short distance in Volunteer State but traverses through two mountain ranges. After Trade, it ventures into Mountain City through a 40 mph (64 km/h) zone while bypassing the downtown area. After Mountain City, begins the two mountain ranges and in between them is the area of Shady Valley. After the second mountain range, it flattens out after the south Holston River heading towards Bristol. US 421 enters Virginia on the Old Dominion side of Bristol. Major highway junctions between the NC and VA state lines are SR-67 in Mountain City, SR-91 the new Mountain City bypass, SR-394, and US 11E.

US 421 enters from Tennessee in Bristol and goes west to Weber City, Duffield, and Pennington Gap in Southwest Virginia. The route goes through the Cumberland and Powell Mountains, and goes by Natural Tunnel State Park near Duffield.

US 421 enters the Bluegrass State from the Pennington Gap area in Virginia into Harlan County. For the distance between the state line and closer to Hal Rogers Parkway, it is a major north-south two lane highway in eastern Kentucky. However, its passing zones are largely limited to a stretch after the Virginia line, through Harlan and along US 119. US 421 then crosses Pine Mountain and enters Leslie County, turning west at Hyden and paralleling the Hal Rogers Parkway, which is formerly Daniel Boone Parkway, which likely precludes any addition of passing zones in the future. Therefore, this is a local road but maintains a 55 mph (89 km/h) speed limit for the most part. Between Manchester and McKee, it functions as more of a local road with many curves. The roadway improves as it descends from the Cumberland Plateau into the Bluegrass Region at Big Hill in Madison County near Berea. It joins US 25 at the Blue Grass Army Depot south of Richmond and roughly parallels I-75 towards Lexington. Through Lexington, it is Richmond Road, Main Street and Leestown Road, collectively the first major thoroughfare in Kentucky. Leestown, on the Kentucky River, was established before the state capital of Frankfort. The segment between Lexington and Frankfort was once designated KY 50, suggesting its importance, but the number was abandoned, perhaps at the time it was made part of US 421.[citation needed] US 62 runs briefly with US 421 in this section. I-64 and US 60 are the four-lane roads connecting Lexington and Frankfort, but US 421 still provides the most direct route. In Frankfort, US 421 runs with US 60 on the west side of town, then runs withn the city' northern bypass, briefly joining US 127. In this section, which runs generally east–west, northbound US 421 runs with southbound US 127 and vice versa. Northbound US 421 traverses the very hilly area northwest of Frankfort, then through Henry and Trimble counties, exiting the state at Milton, crossing the Ohio River into Madison, Indiana. Preliminary construction has begun on a replacement bridge at this location.

US 421 winds through the southern part of Indiana as it runs from Madison, in the southeastern part of the state, to Indianapolis. North of Greensburg, US 421 intersects and merges with I-74 west, through the Shelbyville area en route to Indianapolis. Originally, US 421 followed Southeastern Ave. into downtown Indianapolis, where it merged with US 40 (Washington St.) to West St., then turned north, following West St., Northwestern Ave. (later MLK Dr.), and Michigan Rd. up to the northwest side of the city. US 421 went past the Pyramids, an Indianapolis landmark. North of Indianapolis, US 421 continues and goes in and out of small towns passing SR 10 in San Pierre,Indiana. US 421 ends at its junction with US 20 on the south side of Michigan City. Originally, the highway's end was a few miles north at the junction with US 12 near the shores of Lake Michigan.

US 421 mainly followed the same route between Clinton and Wilmington. However, there are several old alignments from Clinton to the Tennessee state line.