The protective effect of salidroside (SDS) isolated from Rhodiola sachalinensis A. BOR. (Crassulaceae), was investigated in acetaminophen (APAP)-induced hepatic toxicity mouse model in comparison to N-acetylcysteine (NAC). Drug-induced hepatotoxicity was induced by an intraperitoneal (i.p.) injection of 300 mg/kg (sub-lethal dose) of APAP. SDS was given orally to mice at a dose of 50 or 100 mg/kg 2 h before the APAP administration in parallel with NAC. Mice were sacrificed 12 h after the APAP injection to determine aspartate aminotransferase (AST), alanine aminotransferase (ALT), and tumor necrosis factor-alpha (TNF-α) levels in serum and glutathione (GSH) depletion, malondialdehyde (MDA) accumulation, and caspase-3 expression in liver tissues. SDS significantly protected APAP-induced hepatotoxicity for SDS improved mouse survival rates better than NAC against a lethal dose of APAP and significantly blocked not only APAP-induced increases of AST, ALT, and TNF-α but also APAP-induced GSH depletion and MDA accumulation. Histopathological and immunohistochemical analyses also demonstrated that SDS could reduce the appearance of necrosis regions as well as caspase-3 and hypoxia inducible factor-1α (HIF-1α) expression in liver tissue. Our results indicated that SDS protected liver tissue from the APAP-induced oxidative damage via preventing or alleviating intracellular GSH depletion and oxidation damage, which suggested that SDS would be a potential antidote against APAP-induced hepatotoxicity.