Wildlife road mortality is a worldwide problem. In tropical developing countries, it is urgent to implement and test mitigation measures to reduce wildlife-vehicle collisions, especially because financial resources are limited and alternatives should be considered such as retrofitting of existing underpasses. We assessed herein the use of 12 unfenced crossing structures, including drainage culverts and cattle boxes, by medium and large mammals along a highway in the Brazilian Cerrado. Underpass usage was monitored for 3682 trapping nights from September 2017 to May 2018. We recorded 20 species of medium and large wild mammals in a total of 4212 events. The most frequent mammals were lowland tapirs (Tapirus terrestris, n = 1154), followed by capybaras (Hydrochoerus hydrochaeris, n = 910), and crab-eating foxes (Cerdocyon thous, n = 271). Underpass usage did not differ between drainage culverts and cattle boxes, but species composition differed among the structures, suggesting that terrestrial mammals prefer cattle boxes while some semiaquatic species used only culverts. We identified 28 different individual tapirs using the underpasses and we estimated over 180 tons of tapir biomass crossing under the highway. Tapir underpass usage did not differ between cattle boxes and culverts, but it was negatively associated with the presence of pasture area around the structures. Our study suggests that, although underpasses show high mammal usage suggesting a satisfactory level of connectivity, combining this method with fencing would be critical to substantially decrease wildlife road mortality and increase human safety.