Particulate organic matter (POM), dissolved organic matter (DOM), bacteria and cladoceran were sampled seasonally at Zhihugang Estuary and Lake Center in Taihu Lake. The δ13C of the four organic matter fractions showed consistent temporal variation, with heaviest values in summer and lower at other times of the year. The cladoceran δ13C showed a significant correlation with that of POM, reflecting a heavy dietary dependence on POM during the study period. The bacteria became enriched in 13C compared with that of DOM throughout the sampling dates, although no significant relationship was found between the two fractions. δ13C values of POM, cladoceran and bacteria were all negative significantly correlated with oxidation and reduction potential (ORP), and specific conductivity (SpCond). As for δ15N, the seasonal pattern of food web components was variable. The POM δ15N signature exhibited the most enriched isotope ratios during the summer months when dissolved inorganic nitrogen (DIN) nutrients were at their lowest concentrations. The consumption of DIN in summer can explain in part the progressive accumulation of heavy nitrogen isotopes during this period. Spatially, δ13C and δ15N of the food web components were all slightly depleted at Estuary than that at Lake Center during the study period, possibly due to large allochthonous inputs at Zhihugang Estuary. Relatively wide ranges of stable isotopic values from both sites suggest that seasonality should be considered when attempting to establish food web structures in a eutrophic lake.