This chapter on wildlife underpasses on U.S. Highway 64 in North Carolina is from a book on highways, wildlife, and habitat connectivity. U.S. 64 is an important route in North Carolina connecting major population centers and highways that underwent a major upgrade from a two-lane rural road to a major highway. New routes were proposed for a large portion of the project (28 miles) to improve driver safety and increase speed limits to 70 miles per hour (from the previously posted 55 mph). The authors review the geographical, historical, political, and social setting; the roadway and environmental issues; the rationale for the project; critical factors; outcomes of the project; and lessons learned. The area of the project supports high wildlife densities, including American black bears, white-tailed deer, red wolves, and bobcats. Critical factors to be incorporated into wildlife mitigation measures: driver safety, underpass construction costs, and permitting by the U.S. Fish and Wildlife Service and the U.S. Army Corps of Engineers. The U.S. 64 underpasses, completed in 2005, were the first in North Carolina designed specifically for wildlife and according to specifications provided by the North Carolina Wildlife Resources Commission (NCWRC). The authors describe the underpass specifications recommended based on this project, including size, control of public access, fencing, gates, and maintenance (notably vegetation management). The authors conclude that one of the most beneficial outcomes of this project was the fact that, since the completion of the U.S. 64 underpasses, the North Carolina Department of Transportation (NCDOT) routinely considers wildlife passageways for road projects in the state.