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AIM: To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus (HBV).METHODS: HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d. The surface antigen of HBV (HBsAg) and e antigen of HBV (HBeAg) in supatant were determined by ELISA. HBV DNA in supatant, and intracellular covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA) and pregenomic RNA (pgRNA) were quantified by specific real-time polymerase chain reaction (PCR) or reverse transcription (RT)-PCR.RESULTS: Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line, as indicated by the decline of HBsAg (22.67%, t = 5.439,P = 0.0322 and 22.39%, t = 5.376, P = 0.0329, respectively),HBeAg (55.34%, t = 9.859, P = 0.0101 and 43.97%, t = 14.080, P = 0.0050) and HBV DNA (40.75%, t = 4.570, P = 0.0447 and 75.32%, t =14.460, P = 0.0047) in the supatant. Intracellular cccDNA was also markedly reduced by 63.98% ( t= 6.152, P = 0.0254) and 80.83% ( t = 10.270, P =0.0093), and intracellular rcDNA by 34.35% ( t = 4.776,P = 0.0413) and 39.24% ( t = 10.050, P = 0.0097).In contrast, intracellular pgRNA increased by 6.90-fold ( t = 8.941, P = 0.0123) and 3.18-fold ( t = 7.432, P =0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d, respectively.CONCLUSION: Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid, or inhibiting the activity of the viral DNA polymerase.