U.S. Route 64 is the longest numbered route in the U.S. state of North Carolina, running 604 miles (972 km) from the Tennessee state line to the Outer Banks. The route passes through the westernmost municipality in the state, Murphy, and one of the most easternmost municipalities, Manteo, making US 64 a symbolic representation of the phrase "from Murphy to Manteo" which is used to refer to the expanse of the state. The highway is a major east-west route through the central and eastern portion of the state.

US 64 enters North Carolina in Cherokee County, west of Murphy. The highway serves the cities of Hendersonville, Brevard, Rutherfordton, Morganton, Lenoir, Statesville, Lexington, Asheboro, Siler City, Raleigh, Rocky Mount, Tarboro, Williamston, and Manteo.

The segment from Franklin to Highlands is a mountainous two-lane road limited to moderate-sized trucks. Large trucks are routed via Truck US 64 (US 23/441 and US 74) to Sylva, and Asheville.

Within the Raleigh city limits US 64 follows the southern section of the Beltline. In 2006 a major section known as the Knightdale Bypass opened to ease traffic. After it was completed, US 64 became a divided freeway between Cary and Williamston, and carries at least four lanes between Asheboro and Columbia.

US 64 serves as a gateway to the Outer Banks, ending at Nags Head where it meets U.S. Route 158 and NC 12.

US 64 is part of four scenic byways in the state (indicated by a Scenic Byways sign).

Waterfall Byway is an 98-mile (158 km) byway from Murphy to Rosman; it is known for its more than 200 waterfalls that surround the route. US 64 overlaps almost the entire route, except near Murphy, continues along Brasstown Road and Harshaw Road. This byway also connects to the Nantahala Byway (North of Murphy via US 19), Whitewater Way (NC 281 at Sapphire), and Forest Heritage Scenic Byway (NC 215 at Rosman). Tractor-Trailers are prohibited on this road and it is also not recommended for recreational vehicles or buses.

Black Mountain Rag is an 31-mile (50 km) byway centered at Bat Cave that goes north to Black Mountain via NC 9, south to Edneyville via US 64, and east to Lake Lure via US 64/US 74A. It is named after old fiddle tune, the Black Mountain Rag, for its full of twists, turns, ups and downs. Chimney Rock State Park and Hickory Nut Falls are also located along the byway. This byway also connects to Drovers Road byway by taking west on US 74A at Bat Cave. This byway is not recommended for recreational vehicles or buses.

Alligator River Route is an 71-mile (114 km) byway from New Holland to Manteo; it is known for its expansive wetlands, open farmlands, and Lake Mattamuskeet. US 64 overlaps 36 miles (58 km), from Columbia to Manteo. This byway also connects to the Pamlico Scenic Byway on US 264 in Manns Harbor.

Roanoke Voyages Corridor is an 9-mile (14 km) byway on Roanoke Island; it is known for Fort Raleigh National Historic Site (site of the first English settlement in North America), the North Carolina Aquarium, and Roanoke Island Festival Park. US 64 and US 64 Business overlaps the entire route. This byway also connects to the Outer Banks Scenic Byway in Nags Head.

US 64 was established in 1932; from Tennessee, following NC 28 to Old Fort, overlapping with US 70/NC 10 to Statesville, then finally NC 90 to end at Fort Landing, in Tyrrell County. In late 1934, NC 28, NC 10, and NC 90 were dropped along the route. From the 1970s to the early 2000s, US 64 was made a freeway from east of Knightdale to WIlliamston. Most of the original highway became U.S. 64 Alternate. In the early 2000s, it was also placed on a freeway from Plymouth to Columbia.

Originally, US 64 followed Salem Road, north of Apex; near Cary, it followed Chatham Street and Hillsborough Street before meeting NC 54 on Western Boulevard, after which both go into Raleigh via Hillsborough Street. In central Raleigh, US 64 used Salisbury Street, Edenton Street, East Street, and finally New Bern Avenue. The final section followed the current US 64 Business to Wendell and Zebulon. Between 1950-53, US 64 was removed from central Raleigh and followed then US 70 Alternate via Western Boulevard (in Cary) east to Boylan Avenue, then northeast to South Street before ending back on New Bern Avenue.

In 1960, US 64 was placed on a bypass around Wendell; the old route became US 64 Business. In 1965, US 64 was given its modern routing from Apex to US 1; after concurrency, US 64 followed US 1 north around Raleigh to New Bern Avenue. In 1984, US 64 was removed from the northern arc of the beltline, migrating south to the completed southern arc.

The Knightdale Bypass is a six-lane freeway in Wake County, North Carolina that was finished in July 2005. It is also frequently called the "64 Bypass", although it is only signed as such at the intersection of old US-64 (current US-64 business) and I-440. The 11-mile (18 km) freeway bypasses the town of Knightdale, North Carolina, allowing traffic to flow easier in and out of the Raleigh metropolitan area. U.S. Highway 64 and U.S. Highway 264 have been rerouted onto the Knightdale Bypass which traverses from Raleigh's Inner Loop (Interstate 440) to the US 64/264 freeway in Wendell, North Carolina. An interchange with the eastern segment of Raleigh's Outer Loop (Interstate 540) opened in January 2007. This interchange makes the Knightdale Bypass a key connector between Raleigh's two loops.

US 64 and US 264's previous alignment has been given the designation Business US 64; a business route of US 264 was not included. It travels from I-440 in Raleigh eastward through the town of Knightdale, crossing US 64 in Wendell and rejoining it at the business route's eastern terminus in Zebulon. In order for the business route to be a complete loop, it must share a short one-mile (1.6 km) segment with I-440; however, there are no signs that indicate this concurrency.

In Asheboro, a new US 64 By-Pass is planned that will go south around the city, with a new connector (an extension of NC 159 Spur) to the North Carolina Zoo. The proposed 13.7-mile (22.0 km) route will cost an estimated $370 million, scheduled to begin construction in 2014.

On February 20, 2013, NCDOT has submitted a request, to AASHTO, to established a new auxiliary route of Interstate 95, bannered Interstate 495. The proposed 44.99-mile (72.40 km) route will begin at Interstate 440/US 64 Business, in Raleigh, and end at Interstate 95, in Rocky Mount, completely overlapping with US 64.