Since aflatoxin B(1) (AFB(1))-mediated hepatic damage is related to the production of AFB(1)-8,9-epoxide and reactive oxygen species, bioactive compounds having antioxidant potentials are suggested to be capable of reducing AFB(1)-induced toxicity. We previously purified a mixture of flavonoids that we named RCMF (Rhus verniciflua Stokes chloroform-methanol fraction), from a traditional Korean food additive and herbal medicine. RCMF exhibited various biological effects, including antioxidant and antitumor activities. In this study, we examined whether RCMF protects against AFB(1)-induced liver injury using in vitro and in vivo systems. Pretreatment of HepG2 cells with RCMF significantly reduced AFB(1)-stimulated production of ROS and malondialdehyde (MDA) to the control levels. RCMF also prevented the reduction in HepG2 cell viability caused by AFB(1). Oral administration of RCMF to mice significantly suppressed an AFB(1)-induced increase in serum levels of alanine aminotransferase, alkaline phosphatase and lactate dehydrogenase. It also prevented MDA formation and blocked decreases in glutathione levels and superoxide dismutase activities in the livers of AFB(1)-treated mice. In addition, RCMF supplementation prevented an AFB(1) -induced decrease in serum titers of IgA and IgG1. Collectively, these results suggest that RCMF attenuates AFB(1)-mediated damage to the liver, and that this effect is at least partially related to the restoration of antioxidant defense systems and an increase in AFB(1)-GSH conjugate formation.