In California, the southern end of the Santa Cruz Mountains has important ecological connections to both the Gabilan Range to the south and the Diablo Range to the east, facilitating wildlife movement, dispersal, and migration of individuals and species. Within our study area, mountain lion populations exhibit high levels of inbreeding because of extreme isolation caused by roads and development, leading to their designation as a candidate species under the California Endangered Species Act. Thus, protecting and restoring ecological connectivity between areas of core habitat in these mountain ranges is a high priority for regional conservation efforts. This study brought together scientists, connectivity experts, conservation practitioners, and the California Department of Transportation Districts 4 and 5 to conduct an applied study assessing connectivity between these three mountain ranges and identify recommendations for connectivity enhancements within this regionally significant, multi-jurisdictional geography. Our methods included wildlife camera monitoring of existing highway undercrossings at 42 sites, roadkill surveys along five state highways, and habitat suitability and cost surface modeling for a suite of terrestrial mammal focal species. We used our results to identify 19 Connectivity Emphasis Sites (CESs) — specific locations with the most opportunities for reducing wildlife– vehicle collisions and improving connectivity for all wildlife, including fragmentation- sensitive species such as American badger and mountain lion. We determined specific connectivity enhancement measures for each of these locations to improve connectivity for one or more focal species and to reduce wildlife–vehicle collisions. Our final report includes site-specific recommendations as well as crossing infrastructure information sheets to help guide agency implementation of proposed improvements. The results and recommendations from the study were used by Caltrans District 5 to program a Project Initiation Document currently underway, which includes preliminary engineering plans for a wildlife overpass on U.S. Highway 101 at one of the highest priority, critically urgent sites in partnership with local land trusts and other stakeholders.