Road ecology is becoming an increasingly important aspect of conservation biology. Carcasses lying on the road often confront visitors travelling to the Kgalagadi Transfrontier Park between Upington and the Twee Rivieren Rest Camp. This study investigated the species killed, the factors contributing to their deaths, and suggested solutions to curtail these mortalities. Twelve surveys to record mammal and bird road mortalities were conducted on the R360 main road between Upington and Twee Rivieren (261 km) from January to September 2007. One hundred and eighty four carcasses were recorded from 22 species, and the most common taxa killed were the bat-eared fox (n = 47) and spotted eagle owl (n = 10). The road mortality rate on the R360 road was very high, 5.44 mammals and 1.14 birds per 100 km. Birds were predominantly killed in summer. Notably more nocturnal mammals were killed than diurnal and ‘indistinct’ species. A mammal hotspot was identified along the 91 km of road that traversed the Gordonia duneveld. Since the nine roadside traffic warning signs erected on the R360 road had no measurable impact on road mortalities, it is recommend that three rumble strip sections with accompanying signage be erected in the hotspot to slow down vehicles and curtail mortalities.