Nutraceuticals are widely used by the general public, but very little information is available regarding the effects of nutritional agents on drug toxicity. Excessive doses of acetaminophen (APAP, 4-hydroxyacetanilide) induce hepatic centrilobular necrosis. The naturally occurring substance S-adenosyl-l-methionine (SAMe) has been reported to reduce the hepatic toxicity of APAP. The present study was designed to investigate the hepatoprotective effects of SAMe in comparison to the clinically used antidote N-acetylcysteine (NAC). Male C57BL/6 mice were injected intraperitoneally (i.p.) with an equimolar dose (1.25 mmol/kg) of either SAMe or NAC just before APAP, and the groups were denoted SAMe+APAP and NAC+APAP, respectively. Mice were immediately injected i.p. with 300 mg/kg APAP, and hepatotoxicity was evaluated after 4 h. SAMe was more hepatoprotective than NAC at a dose of 1.25 mmol/kg as liver weight was unchanged by APAP injection in the SAMe+APAP group, whereas liver weight was increased in the NAC+APAP group. SAMe was more hepatoprotective for APAP toxicity than NAC, because alanine aminotransferase levels were lower in the SAMe+APAP. Pretreatment with SAMe maintained total hepatic glutathione (GSH) levels higher than NAC pretreatment before APAP, although total hepatic GSH levels were lower in the SAMe+APAP and NAC+APAP groups than the vehicle control values. Oxidative stress was less extensive in the SAMe+APAP group compared with the APAP-treated mice as indicated by Western blots for protein carbonyls and 4-hydroxynonenal-adducted proteins. In summary, SAMe reduced APAP toxicity and was more potent than NAC in reducing APAP hepatotoxicity.