Analyses of mercury (Hg) isotope ratios in fish tissues are used increasingly to infer sources and biogeochemical processes of Hg in natural aquatic ecosystems. Controlled experiments that can couple internal Hg isotope behavior with traditional isotope tracers (δ 13 C, δ 15 N) can improve the applicability of Hg isotopes as natural ecological tracers. In this study, we investigated changes in Hg isotope ratios (δ 202 Hg, Δ 199 Hg) during bioaccumulation of natural diets in the pelagic Pacific bluefin tuna ( Thunnus orientalis ; PBFT). Juvenile PBFT were fed a mixture of natural prey and a dietary supplement (60% Loligo opalescens , 31% Sardinops sagax, 9% gel supplement) in captivity for 2914 days, and white muscle tissues were analyzed for Hg isotope ratios and compared to time in captivity and internal turnover of δ 13 C and δ 15 N. PBFT muscle tissues equilibrated to Hg isotope ratios of the dietary mixture within ∼700 days, after which we observed a cessation in further shifts in Δ 199 Hg, and small but significant negative δ 202 Hg shifts from the dietary mixture. The internal behavior of Δ 199 Hg is consistent with previous fish studies, which showed an absence of Δ 199 Hg fractionation during Hg bioaccumulation. The negative δ 202 Hg shifts can be attributed to either preferential excretion of Hg with higher δ 202 Hg values or individual variability in captive PBFT feeding preferences and/or consumption rates. The overall internal behavior of Hg isotopes is similar to that described for δ 13 C and δ 15 N, though observed Hg turnover was slower compared to carbon and nitrogen. This improved understanding of internal dynamics of Hg isotopes in relation to δ 13 C and δ 15 N enhances the applicability of Hg isotope ratios in fish tissues for tracing Hg sources in natural ecosystems.