Conservation of large carnivores, such as the African lion, requires preservation of extensive core habitat areas, linkages between them, and mitigation of human-wildlife conflict. However, there are few rigorous examples of efforts that prioritized conservation actions for all three of these critical components. We used an empirically optimized resistance surface to calculate resistant kernel and factorial least cost path predictions of population connectivity and conflict risk for lions across the Kavango-Zambezi Transfrontier Conservation Area (KAZA) and surrounding landscape. We mapped and ranked the relative importance of (1) lion dispersal areas outside National Parks, (2) corridors between the key areas, and (3) areas of highest human-lion conflict risk. Spatial prioritization of conservation actions is critical given extensive land use redesignations that are reducing the extent and increasing the fragmentation of lion populations. While our example focuses on lions in southern Africa, it provides a general approach for rigorous, empirically based comprehensive conservation planning based on spatial prioritization.