Abstract Knowledge about the feeding ecology of didelphid marsupials remains incipient, especially in environments with marked seasonality in resource supply, such as in the Cerrado. We analysed the diet composition of Thylamys macrurus in Cerrado patches in central-western Brazil. We also evaluated seasonal and sexual variation in the species’ diet, as well as if the accumulation of fat in the tail shows a seasonal pattern. We obtained 87 captures of 68 T. macrurus specimens and 29 faecal samples. Six categories of food items (Hymenoptera, Isoptera, Coleoptera, Arachnida, Blattodea and seeds) were identified. Arthropods were recorded in all samples and seeds in 10.3%. The species showed seasonal variation in diet, consuming primarily of arthropods throughout the year but incorporating a higher proportion of fruits during the wet season. Males and females also differed in diet composition; males consumed a wider range of items in the dry season. Although more detailed analyses are still needed to better understand the T. macrurus food consumption pattern, our results indicate a possible relationship between dietary variation and local availability of resources, suggesting an opportunistic foraging pattern. The accumulation of fat in the tail also presents a seasonal pattern, with a larger tail diameter in the dry season.