Human infrastructure, particularly highway infrastructure, has proven to be a hazard for the resilience of large felid populations throughout the world. The Yucatan Peninsula is no exception and is presently confronted with the establishment of the “Tren Maya,” a continental railway that will reshape the road network of the peninsula. We describe 12 successful crossings of a 4-lane highway on the peninsula by a female puma (Puma concolor) fitted with a GPS radio-transmitter. The median time between sequential locations was 24 h (range 8–56 h), and the average number of days between crossings was 4.33 (SD = 2.50). Felid behavior and movements in relation to roads may be influenced by reduced human mobility due to the COVID-19 pandemic lockdown. We identified a 3-km stretch of highway where the crossings occurred. We describe the characteristics of the area and recommend building wildlife passage structures appropriate for the site.