Mountain lions (Puma concolor) have been studied extensively throughout their wide geographic distribution in the Western Hemisphere, but most research has been conducted at relatively small spatial scales. We used GPS telemetry data contributed by researchers in 16 study areas throughout California to advance understanding of mountain lion spatial ecology. First, we investigated multi-scale resource selection of mountain lions relative to spatially varying risk of intentional human-caused mortality associated with livestock depredation management. We show that individuals that proactively avoided mortality risk when selecting home ranges were able to relax their avoidance of risk when making resource selection decisions within their home ranges. Our analysis also demonstrates that functional responses in resource selection provide a quantitative link between coarse and fine-scale resource selection decisions made by individual animals. Second, we evaluated movement-based resource selection of subadult mountain lions during dispersal, which is the primary mechanism that maintains gene flow between populations. Our results support the hypothesis that anthropogenic landscape features act as barriers to movement that limit demographic and genetic connectivity between populations, elucidating behavioral mechanisms underlying the observed genetic structure between California mountain lion populations. However, our results also suggest that preservation of even relatively small patches of natural habitat and implementing road-crossing infrastructure should enhance connectivity. Third, we investigated intrinsic, ecological, and anthropogenic factors influencing home range size of mountain lions along a statewide gradient of landscape productivity and human disturbance. Our results indicate that home range size is influenced by the abundance and distribution of prey in relatively undisturbed landscapes, whereas anthropogenic features create fragmentation and barrier effects that influence home range size in developed regions. Our work advances understanding of responses of mountain lions to mortality risk from the leading mortality cause in California, behavioral mechanisms of reduced connectivity between populations, and the influence of natural and anthropogenic factors on space use. Management and conservation informed by empirical research will contribute to promoting long-term viability of mountain lions in southern California and along the central coast, which face challenges similar to those of large carnivores around the globe associated with human-wildlife conflict, habitat loss, and fragmentation.