Mercury (Hg) is readily bioaccumulated in seafood, a common ingredient in indigenous cuisines throughout the world. This study investigates Hg speciation in cooked seafood after gastric and intestinal digestion. The results showed that the removal of Hg by washing was negligible. Additionally, the results of our calculations regarding the mass balance of Hg concentration indicated that cooking reduced Hg mainly by means of volatilization and that Hg2+ was more readily reduced than MeHg. Moreover, cooking lowered the bioaccessibility of Hg in seafood: the reduced percent of bioaccessible Hg2+ after cooking ranged from 2 to 35% (on average, 16%). The corresponding numbers were slightly lower compared with those for MeHg (on average, 19%). Furthermore, there might be a chemical transformation of Hg during in vitro gastrointestinal digestion. The results of in vivo tests in laboratory mice suggested that methylation of Hg mainly took place in the gastric tract, whereas demethylation of Hg occurred primarily during intestinal digestion. These findings indicate that the bioaccessibility of He2+ and MeHg was not only related to their initial concentrations in the food samples, but also that further studies on the mechanisms of Hg demethylation and methylation during gastrointestinal digestion are essential for more realistic risk assessments.