Berberine (BBR) is an isoquinoline alkaloid, reported to have multiple pharmacological functions. However, its effects against CCl4-induced oxidative damage remain poorly studied. Therefore, the present study investigated the protective action of BBR, and its antioxidant mechanisms, against CCl4-induced liver injury in rats. A total of 48 rats were randomly arranged into six groups: Control; model; positive control (PC); BBR low-dose (BL); BBR middle-dose (BM); and BBR high-dose (BH). The BL, BM and BH animals received BBR (5, 10 and 15 mg/kg by weight, respectively) orally for 7 consecutive days. Rats in the PC group were given silymarin (150 mg/kg), and the control and model groups were administered distilled water orally. At the end of the experiment, blood samples and livers were collected. To measure the liver biochemical indices, the reactive oxygen species (ROS) generation and the expression levels of related genes and protein, the following methods were used: An automatic biochemical analyzer; flow cytometry; spectrophotometry; reverse transcription-quantitative PCR; western blotting; and hematoxylin and eosin staining. The results revealed that BBR significantly decreased the serum levels of alanine transaminase, aspartate transaminase and alkaline phosphatase, and increased those of glutathione and superoxide dismutase, but decreased malondialdehyde activity in hepatic tissue, and significantly decreased the reactive oxygen species level in hepatocytes. In hepatic tissue, the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), kelch-like ECH-associated protein 1 (Keap-1), NAD(P)H quinone dehydrogenase 1 (NQO-1), heme oxygenase 1 (HO-1), Bcl-2 and Bcl-xL mRNA, and HO-1 protein were elevated, and the expression of p53 mRNA was decreased, particularly in the BH group (15 mg/kg). In conclusion, BBR exerts a protective action against CCl4-induced acute liver injury in rats via effectively regulating the expression of Nrf2-Keap1-antioxidant responsive element-related genes and proteins, and inhibiting p53 pathway-mediated hepatocyte apoptosis.