Human-induced habitat fragmentation threatens connectivity for populations of wide-ranging species by compromising long distance dispersal. We evaluated movement-based resource selection of dispersing mountain lions (Puma concolor) to identify specific landscape conditions influencing movement decisions and connectivity between populations across the US state of California. Our results elucidate how anthropogenic features inhibit dispersal at the statewide scale, as mountain lions avoided development more strongly as both the area of and building density within developed patches increased. Furthermore, dispersing mountain lions strongly avoided crossing roadways but exhibited relaxed avoidance in areas where vegetative cover was present on both sides of the road. Importantly, mountain lions selected even relatively small patches of natural habitat, which may function as stepping-stones promoting movement through otherwise unsuitable habitat. We provide empirical evidence linking genetic structure suggestive of reduced connectivity to underlying behavioral mechanisms. Understanding behavior during dispersal is fundamental to conserving connectivity between and viability of populations persisting within fragmented landscapes—and to facilitate recolonization of historical ranges.