An overdose of acetaminophen (APAP) causes liver injury in experimental animals and humans. The activation step (formation of reactive metabolite, N-acetyl-p-benzoquinone imine by cytochrome P450 system) and the consequent downstream pathway of oxidative stress, nitrosative stress, and inflammation play an important role in APAP-induced hepatotoxicity. Formulation of APAP with an inhibitor of the activation step would be ideal to prevent accidental and intentional APAP toxicity. Dimethyl sulfoxide (DMSO) is a common colorless, inexpensive solvent, and considered safe in human. We hypothesized that a less hepatotoxic APAP if co-formulated with DMSO. To test this hypothesis, C57BL/6 mice were given toxic dose of APAP (250 mg kg-1 , i.p.) mixed with different doses of DMSO (25, 50, 100, and 200 μl kg-1 ). Six hours after APAP treatment, blood and lives were collected for analysis. In DMSO treated groups, there was dose-dependent decrease in markers of liver injury, alanine aminotransferase, and aspartate aminotransferase. Maximum protection was obtained with 200 μl DMSO kg-1 . DMSO was shown to inhibit the activation step by decreasing the rate of GSH depletion in vivo and inhibiting cytochrome P450 system in vitro. Also the levels of lipid peroxides, nitrate/nitrite, tumor necrosis factor-alpha, and interleukin 1β were decreased significantly. In conclusion, DMSO exerts its protective action by inhibiting the metabolic activation of APAP and thus alleviating the downstream, oxidative stress, nitrosative stress, and inflammation via indirect inhibition. Our findings suggest that replacing the current APAP with APAP/DMSO formulation could prevent accidental and intentional APAP toxicity.