Millions of animals are killed on roads annually due to collisions with vehicles, particularly medium–large mammals. Studies on mammal road-kill flourished in Brazil in the last decade and an assessment of research on road-kill impacts at a country level will help define science-based conservation strategies. In this study, we used the compiled scientific literature to provide the state of knowledge on medium and large-sized mammals killed by road traffic in Brazil, their conservation status, and an approximation of the road-kill magnitude. We reviewed a total of 62 scientific papers that reported road-kill data accounting for 11.817 individuals. Of the 102 species of medium–large mammals found in the IUCN list, more than half (n = 62; 61%) were recorded as road-kill on Brazilian roads. The Carnivora order comprises over a quarter (n = 23; 37%) of the total road-killed species. A total of 9 species (14.5%) were classified as threatened, with a further 10 (16%) as Near Threatened. Over half of the road-killed species (n = 33, 53%) showed declining population trends according to their IUCN conservation status. Our extrapolation of the results for the entire Brazilian paved road network showed that the number of medium–large road-killed mammals can reach almost 9 million yearly (maximum 8.7 million; mean 1.3 million), representing a biomass of more than 10000 tons. The highest roadkill rates were recorded for common generalists and least concern species, although there were also threatened and near threatened species within the top 15 highest road-kill rates. The declining population trends found for most species reflect serious conservation concerns, since there is a lack of information on the mortality effects at population levels. Our results suggest that medium–large mammals are severally affected by road mortality in Brazil. More investigations are needed at local and abundance population levels, in a way that allows the inclusion of road network as an important threat for target species impacted by road-kill in the national territory, in order to develop adequate plans to mitigate those impacts.