Although motorways could affect wildlife species, only few studies have been documented on their effects on mortality and isolation. With 2266 road-killed animals representing 97 species, the results of a study on a motorway section emphasized that traffic considerably affected vertebrate populations (14.5 animals day⁻¹100 km⁻¹). Road-killed animals were mainly mammals (43.2%), with predators also suffering critical impacts (21.7% vertebrates). Rare or endangered species such as the Midwife toad, the Blue throat, the little Horseshoe bat, or the European otter were among the victims. Animal mortality exponentially increased with traffic volume. Mortality reached almost 100% of migrants when no passage existed, and this barrier effect was only reduced when underground passages crossed the road restraining the mortality to 31% of migrants in Field mice and 23% in Common toads, while mortality always exceeded 74% in a road section with fauna ducts. It is reasonable to conclude that traffic severely influenced both wildlife species demography and population exchanges resulting in effective population isolation.