Deforestation is a key driver of biodiversity reduction worldwide and impacts ecosystem functioning, ecological processes, and species behavior. Here, we investigated the effects of deforestation on the diet of puma (Puma concolor) in the Brazilian Atlantic Forest. We characterized puma diets in the largest continuous Atlantic Forest remnant and compiled literature data on their diet across the biome for comparison. We divided localities (N = 16) into two systems, namely, preserved areas (>40% forest cover) and human-modified landscapes (HMLs; <40%), and compared the diets in terms of percentage of occurrence, mean weight of mammalian prey (MWMP), and niche breadth. We classified prey according to their body mass (small- to large-sized) and partitioned the contribution of vertebrates (mammals, birds, reptiles, and fish) and mammalian prey (10 orders). The puma diets varied widely across the Atlantic Forest, with prey size decreasing from preserved areas to HMLs. As deforestation increased, the proportion of small-sized prey also increased, leading to a reduction in the MWMP. Niche breadth varied independently of the context or forest cover. Ungulates, large rodents, and xenarthrans were the main prey for pumas in preserved areas, while small rodents and birds accounted for 55% in HMLs. Ungulate predation increased with forest cover augmentation, with the opposite pattern observed for small rodents. The puma diet parameters in the Atlantic Forest and across Neotropical regions were similar, whereas both differed from that of North America, where large-sized prey was more consumed. Deforestation drove pumas to feed on smaller prey (<1 kg), thus highlighting that the effects of deforestation can be even larger and affect the assemblage trophic structure.