Stable isotope analysis was used to investigate seasonal and spatial variations of the food web structure in a large eutrophic lake ecosystem (Lake Taihu, China). Basal food sources, invertebrates and fish were sampled in two lake regions with different environmental conditions and spatial variations in the isotopic composition of lake food webs were found. Overall, more depleted δ13C and enriched δ15N isotope values of organic matter sources and consumers were found in the phytoplankton-dominated lake region than in the macrophyte-dominated region. Wide seasonal variations in the isotopic ratios were also observed in the lake biota, with a general trend towards enriched δ13C and δ15N values in summer and depleted values in winter. This pattern could be explained by a combination of environmental (e.g., irradiance and nutrient inputs) and biotic (e.g., availability of food sources and plasticity in prey item choice) features. Results of isotope mass balance suggest that macrophytes provide some trophic support in the macrophyte-dominated area, but in both lake regions it is more likely that aquatic food webs are phytoplankton based rather than macrophyte based under eutrophic conditions.