The Nuevo Xcan-Playa del Carmen highway in Quintana Roo, bisects the vegetation corridor connecting two Jaguar Conservation Units (JCUs): Yum Balaam (north) and Sian Ka´an (south). The project´s main goal was to describe differential use of available crossing structures (wildlife underpasses and culverts) by mammals present along this highway. We set 28 camera traps along the 54km stretch of the highway covering wildlife underpasses (10), and culverts such as box culverts (9) and pipes (9) from September 2016 until March 2017. A total of 24 jaguar crossings have been recorded exclusively using wildlife underpasses, including four males and two females. At least 18 other mammal species including five of the target priority species (protected by Mexican law) were documented, all of which were native except for two invasive species. In terms of species using the crossing structures, we identified 13 species using wildlife underpasses, nine using concrete box culverts and 10 using concrete pipes. Wildlife underpasses show higher diversity values (Shannon´s exponential index = 5.8 and Inverse Simpson´s index = 4.66) compared to culverts because they allow bigger species to cross. We recommend more highways along the jaguar´s distribution should develop mitigation measures to allow for wildlife connectivity. Wildlife underpasses, along with retrofitted culverts, could help secure not only the permanence of this species by facilitating the functional connectivity between populations but have positive impacts on other neotropical mammalian fauna as well.