Changes in vegetative cover and land use in the southern part of the Isthmus of Tehuantepec in Oaxaca, Mexico, were identified by analysing satellite images from 2001 and 2014. Fluctuations in population density of mammal species during 2001–2016 in response to these changes were analysed. During 2001–2014 the types of land use that increased in area (per year) were prescribed burning (uncontrolled burning caused by humans) by 105.11 ha, seasonal agricultural plots by 58.14 ha, areas without vegetation by 24.54 ha, and human settlements by 4.13 ha. In the same period, savannahs decreased by 103.94 ha, tropical dry forest by 39.5 ha, secondary forests by 14.46 ha, and human-induced grassland by 0.13 ha per year. The loss of these habitats resulted in low population densities of mammals, including the eastern cottontail Sylvilagus floridanus and the Tehuantepec jackrabbit Lepus flavigularis, which is categorized as Endangered on the IUCN Red List. We recommend establishing a communal ecological reserve in the study area to implement appropriate management strategies for grassland communities and develop a programme of semi-captive breeding to conserve L. flavigularis.