Transportation planners are increasingly incorporating roadway design features to mitigate impacts of highways on wildlife and to increase driver safety. We used camera and track surveys to evaluate wildlife use before and after construction of 3 wildlife underpasses and associated fencing on a new section of United States Highway 64 in Washington County, North Carolina, USA. We recorded 242 occasions of white-tailed deer (Odocoileus virginianus) use of underpass areas before highway construction began. Following completion of the highway, we collected 2,433 photographs of 9 species with deer representing 93% of all crossings. Adjusting for differences in number of monitoring days, white-tailed deer use of underpass areas averaged 6.7 times greater after the new highway and underpasses were completed. We recorded 3,614 wildlife crossings of ≥20 species based on track counts, representing most medium and large mammals known to occur in the area and several reptiles and birds. After completion of the highway, we documented wildlife mortality due to vehicle collisions during a 13-month period and recorded 128 incidences representing ≥24 species. Within fenced highway segments, mortalities were lowest near underpasses and increased with distance from the underpasses. However, we also documented more mortalities in fenced areas compared with unfenced areas. With greater distance from an underpass, animals with smaller home ranges seemed less likely to reach the underpass and instead attempted to climb over or crawl under fencing. Based on collision reports from adjacent highway sections, the new section of United States Highway 64 experienced approximately 58% fewer wildlife mortalities (primarily white-tailed deer), suggesting underpasses and fencing reduced the number of deer–vehicle collisions. Continuous fencing between underpasses may further reduce the number of vehicle collisions for deer but additional design features (e.g., buried fencing) should be considered for other wildlife species.