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Gut content analyses have found that round gobies (Neogobius melanostomus) are highly dependent on dreissenid mussels but stable isotope analysis has often suggested that the dreissenid contribution is lower. However, estimation of dietary contributions with stable isotopes relies on accurate discrimination factors (fractionation factors). To test if discrimination values commonly used in aquatic food web studies are suitable for round gobies, we collected round gobies from Oneida Lake, raised them for 63 days under four different diets (Chironomus plumosus, Mytilus chilensis, Dreissenia spp., Euphausia superba) and measured the change in white muscle δ13C and δ15N. Gobies were also collected throughout Oneida Lake for gut content and stable isotope analysis. Diets changed as round gobies grew, with small round gobies (17-42mm) feeding mostly on cladocera and chironomids, intermediate sized gobies (43-94mm) transitioning from chironomid to dreissenid consumption, and larger gobies (95-120mm) predominantly consuming dreissenids, similar to findings in other studies. Discrimination factors were obtained by fitting a commonly used asymptotic regression equation describing changes in fish δ13C and δ15N as a function of time and diet stable isotope ratios. The discrimination factor determined for δ13C (-0.4‰ ± 0.32, SE) was lower than the “standard” value of 0.4‰, while that of δ15N (4.0‰ ± 0.32, SE) was higher than the standard value of 3.4‰. Turnover rates for both δ13C and δ15N were estimated as 0.02 ‰*day-1. The use of experimentally determined discrimination factors rather than “standard” values resulted in model estimates that agree more closely with the observed increasing importance of dreissenids in gut content of larger gobies. Our results suggest that the importance of dreissenid mussels inferred from stable isotope studies may be underestimated when using standard isotopic discrimination values.