Estuarine transition zones (ETZs) are biogeochemically complex, nutrient-rich environments supporting diverse and productive food webs. They may also be sites of microbial production of methylmercury (MeHg) and bioaccumulation of this neurotoxicant at the base of the food web. However, the environmental drivers controlling these mechanisms are unclear. We studied the pattern of MeHg bioaccumulation in zooplankton along the 200 km ETZ of a large North American estuary, the St. Lawrence Estuary (Québec, Canada). Our approach integrated the dynamic variation in ETZ geochemistry, focusing on MeHg speciation change, alongside ecological factors, including community composition and stable isotopic tracers of diet and habitat. MeHg bioaccumulation decreased with distance downstream along the ETZ, driven by the salinity gradient and traced by the isotopic signature of sulfur in zooplankton. MeHg speciation modeling suggested that complexation to dissolved organic matter may be used as a proxy of the trophic transfer of MeHg to zooplankton. Further, the binding of MeHg to organic matter was reduced by the presence of chloride, thus reducing the trophic transfer of MeHg. We propose a conceptual model for MeHg cycling in ETZ of large estuaries that hypothesizes that higher-level consumers in turbid upstream regions may face heightened risks of MeHg toxicity but that Hg levels diminish drastically as salinity increases.