Increase in the use of crossing structures does not mean a decrease in roadkill numbers

• Roads threaten wildlife by causing roadkill and reducing connectivity. • We analyzed data on roadkills and wildlife crossings usage from 14 years of daily monitoring. • The number of wildlife crossings at mitigation structures increased over time. • Roadkill numbers have increased over the years, even in mitigated segments. • Wildlife crossings structures are likely to fail to reduce roadkill due to absence of proper fencing. Roads significantly impact wildlife by increasing mortality and reducing connectivity. Wildlife crossing structures and associated fencing are used worldwide to increase habitat connectivity and reduce roadkill. We assess the effectiveness of mitigation systems composed of clusters of crossing structures and fences in southeastern Brazil, based on 14 years of data for ground-dwelling species (including medium-to-large mammals, large reptiles and a bird). We analyzed whether crossing and roadkill records changed over the years and if roadkill hotspots were eliminated on segments where mitigation measures were installed. Results showed a significant increase in crossing structure usage over time in all mitigation clusters. However, roadkill persisted in mitigated segments, remained higher than in non-mitigated segments, and roadkill hotspots overlapped all mitigation clusters. These results indicate that crossing structures alone do not sufficiently reduce road mortality without appropriate fencing to prevent road access. Our findings highlight the need for well-designed mitigation strategies, reinforcing that fencing quality and extent are critical for reducing roadkill.