The presence of roads has a significant impact on wildlife conservation, since it is as an important cause of direct mortality of individuals as well as a factor of habitat fragmentation. The LIFE SAFE-CROSSING project (https://life.safe-crossing.eu/) aims to mitigate the impact of roads on three priority species in four European countries: the Marsican brown bear and the Wolf in Italy, the Iberian lynx in Spain and the Brown bear in Greece and Romania. Based on the best practices developed during the previous LIFE STRADE project (www.lifestrade.it) the current project started in September 2018 and will last until August 2023. The main activities of the project are: - Identification and monitoring of the high-risk road segments; - Implementation of actions to prevent animal-vehicle collisions (AVC) and to reduce habitat fragmentation; - Raising awareness of drivers and of policy makers on the risk and prevention of AVC. In order to select the road segments where to implement the prevention actions the “Kernel Density Estimation Plus (KDE+)” method was applied, using over 500 cases of road collisions with large carnivores and with other medium and large sized mammals. We also analyzed the telemetry data of radio-tagged animals in order to identify the road segments with the highest crossing rates. An accurate monitoring of hotspots of road kills has then been made to select the exact locations for the installation of the AVC prevention tools, and we also monitored the number and speed of vehicles on the target roads on 24 hours. On the basis of these activities 27 AVC Prevention System, developed in the LIFE STRADE project, will be installed and 30 km of roads will be equipped with innovative road side active reflectors. To favor habitat connectivity 80 existing underpasses will be readapted to increase their use by wildlife. The innovative “neuromarketing” technique is being experimented in order to produce road signs specifically designed for raising awareness of drivers about the road kill problem and to encourage them to adopt an adequate driving behavior. A specific information campaign is carried out with the local driving schools, and a thematic video- game will be developed to involve and attract young people. The LIFE SAFE-CROSSING project therefore is a tool to face the problems of the impact on roads on biodiversity involving associations and private bodies at international level. The main characteristic of this initiative is to act in different ways against road kills and habitat fragmentation and to provide an example for future cases of replication.