The highway runs for 941 miles (1,514 km) from Fort Fisher, North Carolina, to US 20 in Michigan City, Indiana.

US 421 begins at Fort Fisher, North Carolina, and heads generally to the northwest to Michigan City, Indiana.

US 421 begins near Fort Fisher State Recreation Area and heads through North Carolina's southeastern beaches to Wilmington.

It then heads in a northwesterly direction through Clinton, Dunn, Lillington, Sanford and Siler City to the Piedmont Triad region.

US 421 enters from Tennessee in Bristol and goes west to Weber City, Duffield, and Pennington Gap in Southwest Virginia.

US 421 enters the Bluegrass State running 272.4 miles (438.4 km) from the Pennington Gap area in Virginia into Harlan County.

For the distance between the Virginia state line and closer to Hal Rogers Parkway, it is a major north–south two-lane highway.

US 421 then crosses Pine Mountain and enters Leslie County, turning west at Hyden and paralleling the Hal Rogers Parkway.

The roadway improves as it descends from the Cumberland Plateau into the Bluegrass Region at Big Hill in Madison County near Berea.

It then runs through Henry and Trimble counties, exiting the state at Milton, crossing the Ohio River into Madison, Indiana via the Milton–Madison Bridge.