Animal activity patterns correspond to the individual diel cycle time and is an important attribute of species coexistence in ecological communities. However, activity patterns of most Neotropical species are still poorly understood. Based on an 8-year camera-trapping survey conducted between 2009 and 2017, we evaluated puma (Puma concolor) activity patterns in a human-modified landscape in Southeastern Brazil. Our objectives were to determine the activity pattern of pumas and to verify the influence of main prey species and anthropogenic factors on their behavior. We categorized activity patterns of all assessed species based on the proportion of independent records during night and day times. We tested for sex differences in activity patterns of pumas, and measured their overlap with most consumed prey, people, cattle and domestic dogs. Our results suggested that males engaged in mostly nocturnal behavior while females were active both at night and day hours. Pumas exhibited higher coefficient of overlapping with prey species that were most often included in their diet, suggesting that prey availability might influence puma activity or that pumas opportunistically prey upon species with similar activity pattern. Female pumas seem to be more exposed to anthropogenic threats due to higher activity pattern overlap with people and domestic dogs. Our findings provide insights into puma-prey temporal behavior, highlighting the relevance of intrasexual dissimilarity in the activity patterns of a top predator living in a disturbed landscape.