Many important conservation reserves in Latin America are bisected by major roads. The Guanacaste Conservation Area (GCA) in northwest Costa Rica is a globally important tropical dry forest protected area bisected by the Pan-American Highway. This road is the main international north-south route for vehicle traffic in the Americas, and is in the process of being enlarged to accommodate more international commercial transportation. We investigated spatial and temporal patterns of road crossing and road mortality for vertebrates along the highway within the GCA, and correlates of road-kill hotspots. We also monitored fifteen intermittent stream crossing and drainage culverts under the roadway. Road mortality for mammals was low, and associated with areas that closed canopy forest approached the road. Reptile and amphibian mortality was higher, and associated with temporary water bodies and areas of forest canopy cover over the roadway. Mammals and other vertebrates frequently used culverts to cross under the roadway, but some culverts also experienced some human disturbance. These culverts were used despite there being no wildlife fencing to prevent access to the roadway; natural topography and vegetation of the incised seasonal stream ‘barrancas’ guided animals to these passages. We conclude that culverts and bridges over intermittent stream crossings are critical infrastructure features for maintaining habitat connectivity and reducing mammal road mortality in tropical dry forest. Given that road networks are growing rapidly in extent and traffic volume, there is a need for more research on the location and design of effective crossing structures along roads in Latin America and elsewhere in the tropics.