Mercury (Hg) in fish has attracted public attention for decades, and methylmercury (MeHg) is the predominant form in fish. However, the in vivo MeHg demethylation and its influence on Hg level in fish have not been well-addressed. The present study investigated the in vivo demethylation process in a marine fish (black seabream, Acanthopagrus schlegeli) under dietary MeHg exposure and depuration and quantified the biotransformation and interorgan transportation of MeHg by developing a physiologically based pharmacokinetic (PBPK) model. After exposure, we observed a 2-fold increase of the whole-body inorganic Hg (IHg), indicating the existence of an in vivo demethylation process. The results strongly suggested that the intestine played a predominant role in MeHg demethylation with a significant rate (6.6 ± 1.7 day-1) during exposure, whereas the hepatic demethylation appeared to be an extremely slow (0.011 ± 0.001 day-1) process and could hardly affect the whole-fish Hg level. Moreover, demethylation in the intestine served as an important pathway for MeHg detoxification. Our study also pointed out that in vivo MeHg demethylation could influence Hg level and speciation in fish although food is the major pathway for Hg accumulation. Enhancing in vivo MeHg biotransformation (especially in the intestine) could be a potential key solution in minimizing Hg contamination in fish. The related factors involved in intestinal demethylation deserve more attention in the future.