Among numerous anthropogenic impacts on terrestrial landscapes, expanding transportation networks represent one of the primary challenges to wildlife conservation worldwide. Larger mammals may be particularly vulnerable because of typically low densities, low reproductive rates, and extensive movements. Although numerous studies have been conducted to document impacts of road networks on wildlife, inference has been limited because of experimental design limitations. During the last decade, the North Carolina Department of Transportation (NCDOT) rerouted and upgraded sections of United States Highway 64 between Raleigh and the Outer Banks to a 4-lane, divided highway. A new route was selected for a 24.1-km section in Washington County. The new section of highway included 3 wildlife underpasses with adjacent wildlife fencing to mitigate the effects of the highway on wildlife, particularly American black bears (Ursus americanus). We assessed the short-term impacts of the new highway on spatial ecology, population size, survival, occupancy, and gene flow of black bears. We tested our research hypotheses using a before-after control-impact (BACI) study design. We collected data during 2000–2001 (preconstruction phase) and 2006–2007 (postconstruction phase) in the highway project area and a nearby control area (each approx. 11,000 ha), resulting in 4 groups of data (i.e., pre- or postconstruction study phase, treatment or control area). We captured and radiocollared 57 bears and collected 5,775 hourly locations and 4,998 daily locations. Using mixed-model analysis of variance and logistic regression, we detected no differences in home ranges, movement characteristics, proximity to the highway alignment, or habitat use between the 2 study phases, although minimum detectable effect sizes were large for several tests. However, after completion of the new highway, bears on the treatment area became less inactive in morning, when highway traffic was low, compared with bears on the control area (F1, 43 = 6.05, P = 0.018). We used DNA from hair samples to determine if population size and site occupancy decreased following highway construction. For each study phase, we collected black bear hair from 70 hair snares on each study area during 7 weekly sampling periods and generated genotypes using 10 microsatellite loci. We used the multilocus genotypes to obtain capture histories for 226 different bears and used capture-mark-recapture models to estimate population size. Model-averaged estimates of population size decreased on the treatment area from 87.7 bears before construction to 31.6 bears after construction (64% reduction) and on the control area from 163.6 bears to 108.2 bears (34% reduction). Permutation procedures indicated this reduction was proportionally greater for the treatment area (P = 0.086). We also applied a spatially explicit capture-recapture technique to test our research hypothesis. The model with the most support indicated a greater change in density on the treatment area (69% reduction) compared with the control area (24% reduction). We did not observe a treatment effect based on survival of radiocollared bears. We used bear visits to hair snares as detections in multi-season occupancy models and found that occupancy decreased more on the treatment area (preconstruction: Ψ = 0.84; postconstruction: Ψ = 0.44; 48% decline) than the control area (preconstruction: Ψ = 0.91; postconstruction: Ψ = 0.81; 11% decline), primarily as a function of a greater probability of site extinctions (ε) on the treatment area (ε = 0.57) than the control area (ε = 0.17). Finally, individual- and population-based analyses of contemporary gene flow did not indicate the highway was a barrier to movements. Black bear use of the 3 wildlife underpasses was infrequent (17 verified crossings based on remote cameras, track surveys, and telemetry). Only 4 of 8 bears with home ranges near the highway were documented crossing the highway (n = 36 crossings), of which 2 were killed in vehicle collisions. Six additional bears were killed in vehicle collisions from May 2007 to November 2008, after we completed field work. Harvest data indicated that hunting mortality alone could explain the population decline on the control area. On the treatment area, however, hunting mortality only accounted for an approximately 40% population decline; the additional 30% decline we observed likely was caused by other mortality. We speculate vehicle collisions were primarily responsible. We conclude that impacts of the new highway on resident black bears occurred at the population level, rather than the individual or genetic level, but that the impact was smaller than harvest mortality. Increased activity by remaining bears when traffic volumes were low indicated behavioral plasticity. Bear use of the underpasses seemed sufficient to maintain gene flow between areas north and south of the new highway. Effectiveness of wildlife underpasses to reduce mortality of black bears may be enhanced if mitigation includes continuous fencing between crossing structures. For small, isolated populations of threatened or endangered large mammals, the potential demographic impacts of highways are an essential consideration in the transportation planning process. Control of mortality factors and maintaining demographic connectivity are particularly important.