We evaluated puma (Puma concolor) feeding patterns in southeastern Brazilian agricultural landscapes using carbon and nitrogen stable isotope analyses of hair collected from fecal samples (N = 64). We classified the samples into three groups: feeding patterns based on forest remnants, on the agricultural matrix or both. We observed a predominance of consumption of C₃ prey (~47% of individuals) in the area with the highest proportion of forest coverage. Conversely, C₄ prey were highly consumed (~40% of individuals) where the agricultural matrix was predominant. The δ¹³C values for pumas in both areas indicated that their food resources come from both forest remnants and the agricultural matrix and that some individuals preferentially consumed C₄ prey, indicating that food resources from the agricultural matrix make up most of their diet (~46% of prey individuals). The wide range of puma's δ¹⁵N values in both areas indicated a diet based on different types of prey. However, the C₄ group had higher values, indicating that both pumas and their prey feed on enriched resources from the agricultural matrix. The results confirm the high behavioral plasticity of pumas in using highly anthropogenic habitats. The stable isotope analyses conducted in this study yielded new information on large carnivore trophic ecology that might be useful in the development of new conservation strategies in disturbed areas.