It is clear that the numbers of species and individuals are key features of a food web. Still, very few studies investigate how species richness influences trophic diversity. We conducted a natural experiment to examine this question, using streams as ecosystem models, and taking advantage of the sites’ location along the upstream downstream gradient (UDG) within a river catchment. The UDG is known to increase species richness and its effects on trophic diversity are still poorly documented. Using stable isotopes we determined the relative contributions of both species richness and the UDG to fish trophic diversity at two levels of organisation. At assemblage level we measured the isotopic space area occupied by fishes, and at species level we calculated the isotopic niche overlap among species. As expected we found that species richness increased from upstream to downstream. At assemblage level the isotopic space area decreased significantly at sites with higher species richness and increased significantly from upstream to downstream. Conversely the analysis at species level showed that the niche overlap increased with species richness and decreased with the UDG. Taken together these results revealed that the effect of species richness was related to an opportunistic feeding strategy, while the effect of the UDG was due to a diet specialisation of the fish. Both analyses (isotopic space and niche overlap) appear useful in elucidating the mechanisms underlying the observed pattern of trophic diversity.