Roads cause several environmental impacts, such as roadkill of wild animals, which may result in the decrease of local fauna density. Roadkill occurs because roads intersect the habitats of many taxa, especially in fragmented landscapes, such as the Cerrado biome. The present study aimed to determine whether demographic variables or biological characteristics influence the mammal's roadkill rate. A survey was conducted in the section of the GO-060 highway located between the municipalities of Trindade and Iporá, from April to September 2012, during the dry season. We used Kruskal-Wallis tests to investigate possible differences among roadkills per sector, species, order, food habit, and activity pattern. Pearson correlation was used to verify the association between roadkill rate and home range. The influence of body mass on the roadkill rate was estimated through a simple linear regression. To verify whether human demographic variables influence the roadkill, we used a multiple linear regression. We recorded 89 individuals of mammals during 18 field trips and obtained a roadkill rate of 0.025 individuals/km/day. Mammals with different food habits have different roadkill rates, once herbivorous mammals are less prone to be roadkilled than omnivorous and insectivorous species. Characteristics such as home range, body mass and activity patterns were not determinant of roadkill rates in this study. Similarly, human demographic variables did not influence the number of roadkills. Biological and ecological characteristics should be considered when implementing measures to reduce roadkill. Hence, it becomes clear that surveys of fauna roadkill should consider both human demographic and biological variables, once, in this research, none of them are relevant by itself, they can be considered complementary information, and can be used to support practical actions.