Lotic environments show a longitudinal gradient related to physical and chemical features that consequently can affect the metabolism and diversity of the aquatic biota. Variations on both the exposure to sunlight and input of allochthonous material are partially responsible for this gradient. We investigated the trophic ecology of the insectivore fish Characidium vidali Travassos 1967 (Characiformes; Crenuchidae) along a mid-sized river in the Atlantic Forest in Southeastern Brazil (Macaé river). We predicted that autochthonous basal resources are more important than allochthonous resources for C. vidali occurring in downstream sites, and that autochthonous resources are mor important in the site further upstream. We analyzed the diet (gut content) and assimilation (stable isotopes) of food resources this fish species. A total of 36 food items were found in stomach contents, all of autochthonous origin. Aquatic insect larvae (especially Chironomidae, Simuliidae e Baetidae) were the most consumed items. Mixing models using the diet as prior information confirmed Chironomidae and Ephemeroptera as the most assimilated prey in all sites, but also revealed the importance of Trichoptera for the diet of C. vidali. Mixing models also showed that over 70% of carbon assimilation by C. vidali and its prey comes from periphyton in all sites, contradicting expectations that allochthonous resources play a more important role in upstream food webs.