The rapidly-growing urban-wildland interface represents a frontier of human-wildlife conflict worldwide. Where natural and developed areas meet, there is potential for negative interactions between humans and wild animals, including wildlife-vehicle collisions. Understanding the environmental and anthropogenic factors associated with these collisions can inform transportation and habitat planning, ultimately reducing animal mortality and human costs. In this study, we investigated the effects of land cover, fencing, lighting, and traffic on the location, species, and abundance of roadkill on the well-trafficked Interstate 280 in California. The highway is situated just south of San Francisco, between a large wildlife refuge to the west and dense residential areas to the east, presenting a major barrier to wildlife movement and providing a unique urban study area with threats to habitat connectivity. We used linear regression models to identify factors that were correlated with overall and species-specific roadkill incidence. Areas with more development on the urban side of I-280 were associated with higher amounts of overall roadkill. Our findings suggest that most animals are crossing between the wildlands on the west side of the highway and urban areas on the east side. We found this pattern to be especially strong for raccoon (Procyon lotor) and black-tailed deer (Odocoileus hemionus). The presence of lighting corresponded to higher incidence of roadkill for deer and raccoons, and for all species combined. We also found strong evidence for seasonality of roadkill incidence, which most likely correlates to differences in movement and dispersal across life history stages.