AbstractIn Tanzania, a proposed road through the Serengeti National Park has caused international debate as to whether it will cause irreversible damage to the ecosystem. We assessed the effect of current roads and traffic in the Serengeti ecosystem on a model ungulate species, the impala antelope (Aepyceros melampus). We measured how faecal glucocorticoid metabolites (FGM), an indicator of physiological stress levels, were affected by different levels of traffic while accounting for other stress‐related variables such as distance to roads, vegetation type and group size. A total of 196 faecal samples were collected. FGM were measured using an enzyme immunoassay (EIA) validated for several ruminants. FGM levels were significantly elevated near major roads with higher traffic levels compared to roads with less traffic. Predators and illegal hunting might also affect FGM levels in the impala, but the potential effect of these variables was inconsistent with the recorded FGM levels and cannot explain the observed variation. These results show for the first time that an African mammal exhibits significant physiological stress in relation to roads and traffic.