Chronic arsenic exposures via the consumption of contaminated drinking water are clearly associated with many deleterious health outcomes, including anemia. Following exposure, trivalent inorganic arsenic (As III ) is methylated through a series of arsenic (+III oxidation state) methyltransferase (As3MT)-dependent reactions, resulting in the production of several intermediates with greater toxicity than the parent inorganic arsenicals. The extent to which inorganic vs. methylated arsenicals contribute to As III -induced hematotoxicity remains unknown. In this study, the contribution of As3MT-dependent biotransformation to the development of anemia was evaluated in male As3mt -knockout (KO) and wild-type, C57BL/6J, mice following 60-day drinking water exposures to 1 mg/L (ppm) As III . The evaluation of hematological indicators of anemia revealed significant reductions in red blood cell counts, hemoglobin levels, and hematocrit in As III -exposed wild-type mice as compared to unexposed controls. No such changes in the blood of As3mt -KO mice were detected. Compared with unexposed controls, the percentages of mature RBCs in the bone marrow and spleen (measured by flow cytometry) were significantly reduced in the bone marrow of As III -exposed wild-type, but not As3mt -KO mice. This was accompanied by increased levels of mature RBCS in the spleen and elevated levels of circulating erythropoietin in the serum of As III -exposed wild-type, but not As3mt -KO mice. Taken together, the findings from the present study suggest that As3MT-dependent biotransformation has an essential role in mediating the hematotoxicity of As III following drinking water exposures.