Objective: The aim of this study was to study the effect of Ginkgo biloba extract (EGb761) on metabolism of aflatoxin B1 (AFB1) in Wistar rats. Methods: Seventy one Wistar rats were assigned at random to groups A, B and C. Rats in groups A, B were injected with AFB1 (intraperitoneal, 100–200 μg/kg body weight, 1–3 times/week). Group C was normal control. Rats in group B were fed in food with EGb761, while rats in groups A, C were given normal food. Blood samples were collected and liver biopsies were performed on the 14th, 28th and 42nd week. All the rats were sacrificed on the 64th week. The incidence of hepatocarcinoma was investigated. The hepatic phase I drug-metabolizing enzyme Cytochrome-P450 (CYP450) and phase II metabolizing enzyme glutathione S-transferase (GST) were analyzed with spectrometry. Serum AFB1-lysine adduct levels were assessed with high performance liquid chromatography (HPLC). The expression of 8-hydroxydeoxy-guanosine (8-OHdG) was measured with immunohistochemistry. Results: The incidence of hepatocellular carcinoma (HCC) in group B was significantly lower than that in group A (26.92% vs 76.00%, P < 0.001). No HCC developed in group C. EGb761 showed no effects on the activities of CYP450 and GST in rat liver tissues. The level of AFB1-lysine adduct reached the peak (4356.01 pg/mg albumin) at the 14th week in group A. EGb761 significantly inhibited the formation of AFB1-lysine adduct in serum by 13.07% at the 14th week (P = 0.033), and 73.63% at the 42nd week (P = 0.002). The expression of 8-OHdG protein in rat liver tissues in group B was significantly lower than that in group A at the 28th, 42nd, and 64th week (P < 0.05). Conclu-sion: The main mechanism underlying the effect of EGb761 in blocking hepatocarcinogenesis induced by AFB1 may not be fully attributable to its influence on the activity of liver phase I and phase II metabolizing enzymes. EGb761 inhibits the produc-tion of AFB1-lysine adducts, decreases the expression of 8-OHdG protein, and finally alleviates the DNA oxidative injury, which may be one of the mechanisms for the effects of EGb761 in inhibiting or delaying AFB1-induced hepatocarcinogenesis.