The giant anteater (Myrmecophaga tridactyla) is an iconic South American mammal, listed as Vulnerable to extinction by the International Union for Conservation of Nature. They are among the most affected species by wildlife vehicle collision (WVC) in the state of Mato Grosso do Sul (MS), Brazil. Therefore, between 2017 and 2020, we conducted the first phase of the Anteaters & Highways Project to monitor wildlife carcasses along highways as well as monitor wild anteaters living near roads. Results allowed us to quantify and evaluate the impact of road mortality on populations, investigate giant anteater movement patterns, and barrier effects. Mitigation measures are key for reducing road-related negative effects on biodiversity, reducing economic impacts from material damages and increasing traffic safety for humans and animals. We recorded 12.400 roadkill in 1.158 km of highways, including 762 giant anteaters. Our results showed that vehicular collisions decrease population growth rate of giant anteaters by half, posing a serious threat to the species’ long-term survival. About 40% of roadkilled animals can cause significant material damage to vehicles and preventing collisions by fencing the road on hotspots of mortality can offset these investments in 9-25 years. Furthermore, interviews with 229 truck drivers in MS showed that most collisions are not intentional. To deviate or to brake can cause an accident, so due to safety concerns, drivers unfortunately resort to hitting the animal. Since there is little room to influence driver behavior when he/she encounters an animal on the road, one of the strategies is to reduce the rate of encounters with these animals by decreasing driving in hotspots at night, during which 80-90% of accidents occur. The WVC mitigation process, although beneficial, is a complex subject involving several institutions and decision makers with different interests and goals. In the second phase of the project, we aim to reduce WVC through the involvement of various sectors of society, using the giant anteater as the ambassador for safer roads. This is an innovative and experimental project that will apply the expertise from phase one and use creative approaches to achieve 7 new objectives to reduce WVC. From 2021 to 2023, we will work to transform the state of Mato Grosso do Sul into a reference for mitigation of WVC through the involvement of different stakeholders (authorities, drivers, highway workers, truck drivers, transportation companies, civil society, schools). Research findings will be used to develop landscape and road management guidelines to mitigate the impact wildlife vehicle collisions on biodiversity and human safety in the Cerrado and Pantanal biomes.