Purpose: Glehnia littoralis has been used to treat ischemic stroke, phlegm, cough, systemic paralysis, antipyretics and neuralgia. The pharmacological mechanisms of Glehnia littoralis include calcium channel block, coumarin derivatives, anticoagulation, anti-convulsive effect, as well as anti-oxidant and anti-inflammatory effects. Alpha-amanitin (α-amanitin) is a major toxin from extremely poisonous Amanita fungi. Oxidative stress, which may contribute to severe hepatotoxicity was induced by α-amanitin. The aim of this study was to investigate whether Glehnia littoralis ethyl acetate extract (GLEA) has the protective antioxidant effects on α-amanitin -induced hepatotoxicity. Methods: Human hepatoma cell line HepG2 cells were pretreated in the presence or absence of GLEA (50, 100 and 200μg/ml) for 4 hours, then exposed to 60μmol/L ofα-amanitin for an additional 4 hours. Cell viability was evaluated using the MTT method. AST, ALT, and LDH production in a culture medium and intracellular MDA, GSH, and SOD levels were determined. Results: GLEA (50, 100 and 200μg/ml) significantly increased the relative cell viability by 7.11, 9.87, and 14.39%, respectively, and reduced the level of ALT by 10.39%, 34.27%, and 52.14%, AST by 9.89%, 15.16%, and 32.84%, as well as LDH by 15.86%, 22.98%, and 24.32% in culture medium, respectively. GLEA could also remarkably decrease the level of MDA and increase the content of GSH and SOD in the HepG2 cells. Conclusion: In the in vitro model, Glehnia littoralis was effective in limiting hepatic injury afterα-amanitin poisoning. Its antioxidant effect is attenuated by antidotal therapy.