The Gaviota Coast, located in one of the last undeveloped areas in coastal southern California and within an important ecoregional transitional zone, encompasses coastal terraces, bluffs, canyons, rugged slopes, and lowlands within the foothills of the Santa Ynez Mountains. The area is a biodiversity hotspot containing grassland, chaparral, oak woodland, riparian, and estuarine habitats home to over 50 special-status species. U.S. 101 passing through the Gaviota Coast is a four-lane highway that bisects large blocks of intact, native habitat. The area has a history of wildlife-vehicle collisions (WVCs) including species such as the imperiled mountain lion, raising community concerns about wildlife-roadway conflicts. In response to community concerns, Caltrans District 5 initiated a Wildlife Connectivity Assessment (WCA) to better understand existing wildlife connectivity conditions and to develop options for improving wildlife connectivity and reducing WVCs throughout the region. The purpose of the WCA was to provide a data-driven assessment to identify wildlife-highway conflict areas, areas of successful wildlife movement, and opportunities for improving connectivity and reducing WVCs. As part of the WCA process Caltrans convened a diverse and inclusive group of partners, stakeholders, and community members and gathered regularly to exchange information about the WCA and collective goals. The WCA specifically gathered detailed information to assess physical and biological conditions of the region at both landscape and local scales. The WCA also included wildlife passage assessments of culverts and bridges, a wildlife camera study to document wildlife passages at culverts and bridges, and a systematic roadkill study. An independent panel of subject-matter experts was also convened to review the study methodology. The information and data collected were used to determine the existing conditions of wildlife connectivity and roadway conflict in the area and identify connectivity enhancement opportunities. A wildlife connectivity mitigation toolbox was developed to determine a range of mitigation options. Next, feasibility assessments of the mitigation options were conducted, resulting in a final list of options deemed suitable for the study area. Recommendations were further developed on the implementation of the connectivity enhancement tools. Guidance on action planning, implementation, funding, and community and stakeholder inclusion were also developed. This project provides a valuable case study on a robust and methodical approach to conducting WCAs, engaging partners, stakeholders, and communities, developing feasible mitigation recommendations, and supporting early planning. This approach will help safeguard future funding of mitigation and support the project’s goals of improving wildlife connectivity, reducing the risk of WVCs, supporting wildlife conservation, and improving public safety in the Gaviota Coast.