A deep investigation on the trophic structure of a fish assemblage in a reference condition river is important since allow the comparison and determination of the extent in which other sites are degraded. It is especially relevant when we consider a reference condition river located in a basin under influence of several anthropogenic activities, as is the case of the Rio Cipo, an important tributary of the polluted Rio das Velhas Basin, Brazil. We aimed to characterize the trophic structure of the fish assemblage of the Rio Cipo using the carbon (delta C-13) and nitrogen (delta N-15) stable isotopes of fish and several available food resources sampled at two sites along the Rio Cipo in the dry and wet seasons. We found that the delta N-15 of fishes change among trophic guilds, but not change between sites and seasons. The delta C-13 values in turn, varies among trophic guilds and between seasons, and the changes between sites are not very expressive. Detritivorous fish presented the greatest variation in delta C-13 values. Although all detritivorous species appeared to consume the same basal resources (algae, periphyton, POM and CPOM), the proportion of assimilation of each resource varied among them. Two individuals ofMyleus micans(herbivorous) and two ofPiabarchus stramineus(omnivorous), were found to differ substantially within the food web by presenting the most enriched values of delta N-15 and delta C-13 in the wet season at the site close to the confluence with other rivers. These outliers had influence in the broad isotopic niches of herbivorous and omnivorous fish. This result suggest that these species have a large spatial range and may be feeding in places with resources enriched in delta N-15 and delta C-13, outside of the Rio Cipo, as well as highlights the importance of considering the influence of downstream polluted areas on the trophic structure of this reference condition river.