Stable sulphur isotopic composition (δ34S) of gizzard shad Dorosoma cepedianum was used to investigate the seasonal and ontogenetic variation in the diet of young and adult fish. This study evaluated fish from a hypereutrophic lake that had recently undergone a 40% reduction of large (>300 mm total length, LT) D. cepedianum biomass as part of a biomanipulation experiment, which aimed at reducing internal nutrient loading. Dorosoma cepedianum δ34S values showed evidence of ontogenetic changes with young fish (<200 mm LT) depending more on benthic food sources than adults (>200 mm LT). The δ34S composition of the adult fish suggested an increasing importance of zooplankton in the diet, although benthic food sources remained part of the diet of all D. cepedianum collected in this study. The results indicated that benthic feeding is used by D. cepedianum of all sizes, suggesting that biomanipulation efforts may need to target all sizes of fish to realize benefits.