Rapid increases in urban land use extent across the globe are creating challenges for many wildlife species. Urban landscapes present a novel environment for many species, yet our understanding of wildlife behavioural adaptations to urban environments is still poor. This study compared the vigilance behaviour of a large mammal in response to urbanisation at a landscape level. Here, we investigate urban (n = 12) and non-urban (n = 12) populations of kangaroos in two regions of Australia, and the relationship between kangaroo vigilance and urbanisation. We used a linear modelling approach to determine whether anti-predator vigilance and the number of vigilant acts performed were influenced by land use type (i.e., urban or non-urban), human population densities, kangaroo demographics, and environmental factors. Kangaroo behaviour differed between the two study regions; kangaroo vigilance was higher in urban than non-urban sites in the southern region, which also had the highest human population densities, however no effect of land use was found in the northern region. Season and sex influenced the vigilance levels across both regions, with higher levels seen in winter and female kangaroos. This study is the first to compare urban and non-urban vigilance of large mammals at a landscape level and provide novel insights into behavioural adaptations of large mammals to urban environments.