<p>The trophic structure of the fish assemblage in the Volta Grande reach of the Xingu River, Brazil, was investigated 4 years after the Belo Monte Hydroelectric Power Plant, which has reduced flows in the reach, commenced operation. Fishes were surveyed during four phases of the annual hydrological cycle. We used stable isotope analysis of carbon and nitrogen to infer variation in fish trophic ecology and the contributions of basal producers to fish biomass. We observed that hydrological variation was associated with subtle changes in the isotopic composition of fishes and aquatic and terrestrial sources of basal production. The sizes of the isotopic spaces occupied by fish trophic guilds differed among hydrologic phases, as did the isotopic overlap among trophic guilds. Recent reductions in the magnitude and duration of the annual flood pulse and lateral habitat connectivity have apparently reduced the availability of certain food sources, including allochthonous resources that are seasonally available in floodplain habitats. Given that many fish species inhabit the middle Xingu River, recent changes to its flow regime and ecological dynamics will have long-term consequences for the conservation of its biodiversity, as well as fisheries production, which are important for local food security and livelihoods.</p>