Linear infrastructures represent one of the most important human impacts on natural habitats and exert several effects on mammal populations. Motorways are recognized as a major cause of habitat fragmentation and degradation and of biodiversity loss. However, it is unknown whether motorways lead to increased physiological stress reactions in wild animal populations. We analysed faecal corticosterone metabolites (FCM) in wild populations of wood mice (Apodemus sylvaticus) living in a well-preserved Mediterranean agro-pastoral woodland at different distances (verge, 500 m and 1000 m) from the AP-51 motorway in Spain. Wood mice were captured with Sherman live traps, and fresh faecal samples from 424 individuals were collected and analyzed in the laboratory. The quantification of FCM was performed by a 5α-pregnane-3β,11β, 21-triol-20-one enzyme immunoassay. Results showed that females had higher FCM levels than males, and these levels were higher in breeding females. In addition, FCM levels were positively correlated with body weight of individuals. Wood mice captured where cattle were present showed higher FCM levels than individuals living where cattle were not detected. FCM levels were higher in non-breeding individuals living close to the motorway compared with FCM levels in those individuals captured further from the motorway. This is the first study showing evidence of the motorways' impact on physiological stress reactions in wild wood mice populations. Understanding how free-living animals are influenced by human interventions could help to understand other subtle changes observed in wild animal populations. Since mice are used world-wide as research models these results could open new perspectives testing human influence on the natural environment and trade-offs of species in degraded ecosystems.