Understanding the carbon sources supporting aquatic consumers in large rivers is essential for the protection of ecological integrity and for wildlife management. The relative importance of terrestrial and algal carbon to the aquatic food webs is still under intensive debate. The Yangtze River is the largest river in China and the third longest river in the world. The completion of the Three Gorges Dam (TGD) in 2003 has significantly altered the hydrological regime of the middle Yangtze River, but its immediate impact on carbon sources supporting the river food web is unknown. In this study, potential production sources from riparian and the main river channel, and selected aquatic consumers (invertebrates and fish) at an upstream constricted-channel site (Luoqi), a midstream estuarine site (Huanghua) and a near dam limnetic site (Maoping) of the TGD were collected for stable isotope (δ13C and δ15N) and IsoSource analyses. Model estimates indicated that terrestrial plants were the dominant carbon sources supporting the consumer taxa at the three study sites. Algal production appeared to play a supplemental role in supporting consumer production. The contribution from C4 plants was more important than that of C3 plants at the upstream site while C3 plants were the more important carbon source to the consumers at the two impacted sites (Huanghua and Maoping), particularly at the midstream site. There was no trend of increase in the contribution of autochthonous production from the upstream to the downstream sites as the flow rate decreased dramatically along the main river channel due to the construction of TGD. Our findings, along with recent studies in rivers and lakes, are contradictory to studies that demonstrate the importance of algal carbon in the aquatic food web. Differences in system geomorphology, hydrology, habitat heterogeneity, and land use may account for these contradictory findings reported in various studies.