Responses of trophic structure to reservoir regulation are important to manage and conserve the river ecosystem influenced by dams. We used stable isotope-derived community-wide metrics to describe spatiotemporal variation in trophic niches and trophic structures of fish assemblages in a tributary of the Three Gorges Reservoir (TGR) in China where water level regulation is put into operation annually. A longitudinally increasing trophic space was discovered in this tributary especially when the water level was low. Trophic diversity declined and trophic redundancy increased from the non-flood period to flood season at the upper backwater region. However, trophic space was more stable in the estuarine site. The site differences were associated with availability of food resource and pathway of consumers, as specifically, disturbance derived from flow pulse at the end of backwater site altered diet composition of the fish consumers, while immense water body in the lower confluence area limited the flood influences as well as promoted exploitation of diverse available sources. Moreover, the declined water level control facilitated the contraction of the trophic niche size at the tail backwater site because of remarkable riverine morphology alternation leading to decreasing water body whereas the rising water level regulation homogenized trophic structure along the downstream river. Our study revealed that there was different response of fish assemblages along longitudinal fluvial gradient to the water level regulation in the reservoir and targeted management and conservation measures are needed for the different sections of the tributary.