Identifying metabolism and detoxification mechanisms of Hg in biota has important implications for biomonitoring, ecotoxicology, and food safety. Compared to marine mammals and waterbirds, detoxification of MeHg in fish is understudied. Here, we investigated Hg detoxification in Atlantic bluefin tuna Thunnus thynnus using organ-specific Hg and Se speciation data, stable Hg isotope signatures, and Hg and Se particle measurements in multiple tissues. Our results provide evidence for in vivo demethylation and biomineralization of HgSe particles, particularly in spleen and kidney. We observed a maximum range of 1.83‰ for δ202Hg between spleen and lean muscle, whereas Δ199Hg values were similar across all tissues. Mean percent methylmercury ranged from 8% in spleen to 90% in lean muscle. The particulate masses of Hg and Se were higher in spleen and kidney (Hg: 61% and 59%, Se: 12% and 6%, respectively) compared to muscle (Hg: 2%, Se: 0.05%). Our data supports the hypothesis of an organ-specific, two-step detoxification of methylmercury in wild marine fish, consisting of demethylation and biomineralization, like reported for waterbirds. While mass dependent fractionation signatures were highly organ specific, stable mass independent fractionation signatures across all tissues make them potential candidates for source apportionment studies of Hg using ABFT.