Objective：To study the effect of cytotoxic chemotherapy drug epirubicin on hepatitis B virus（HBV） replication in vitro. Methods：The cell viability after epirubicin treatment was detected by MTT assay. The epirubicin was added into the culture of two stable HBV-expression cell lines（HepG2.2.15 cells and HepG2-HBV1.1 cells）. The HBV replication intermediates were extracted from the cytoplasma and supernatant，and then HBV DNA copies were detected by quantitative fluorescent PCR and Southern blot. Results：HepG2.2.15 cells were treated by 0，0.1，0.2，0.5 μmol/L epirubicin respectively，and the intracellular HBV DNA copies（×106） were 3.53±0.26，5.39±0.23，9.40±0.92，19.07±1.47；there were significant differences among these groups（F=10.984，P=0.005）. Similarly，HepG2-HBV1.1 cells were treated by 0，0.1，0.2，0.5 μmol/L epirubicin respectively，and the intracellular HBV DNA copies（×106） were 2.80±0.37，5.14 ± 0.49，14.24±1.29，26.59±1.98；there were significant differences among these groups（F=13.122，P=0.003）. The Southern blot showed that the relative volume of HBV DNA replicative intermediate in HepG2.2.15 cells were 1.00±0.01 and 1.53±0.05 in control group and drug group respectively；there were significant differences between two groups（t=19.202，P=0.002）. Similarly，the relative volume of HBV DNA replicative intermediate in HepG2-HBV1.1 cells were 1.01±0.02 and 3.12±0.20，there were significant differences between two groups（t=18.034，P=0.003）. These results suggested that epirubicin promoted intracellular replication of HBV. Meanwhile，HepG2.2.15 cells were treated by 0，0.1，0.2，0.5 μmol/L epirubicin respectively，and the HBV DNA copies（×106） in the supernatant were 2.37±0.22，3.50±0.27，6.02±0.56，17.74±0.96，there were significant differences among these groups（F=20.726，P=0.001）. Similarly，HepG2-HBV1.1 cells were treated by 0，0.1，0.2，0.5 μmol/L epirubicin respectively，and the HBV DNA copies（× 106） in the supernatant were 3.75±0.35，6.05±0.48，10.28±1.01，20.86±1.81，there were significant differences among these groups（F=14.761，P=0.002）. The Southern blot showed that the relative amount of HBV DNA replicative intermediate in the supernatant of HepG2.2.15 cells were 1.08±0.02 and 2.69±0.05 in control group and drug group respectively；there were significant differences between two groups（t=58.33，P=0.000）. Similarly，the relative amount of HBV DNA replicative intermediate in the supernatant of HepG2-HBV1.1 cells were 1.03±0.01 and 3.52±0.05 in control group and drug group respectively，there were significant differences between two groups（t=85.801，P=0.000）. The results indicated that epirubicin promoted cells secreting or releasing HBV particles. Conclusion：Epirubicin has a direct role in promoting HBV replication，which may be a new mechanism of HBV reactivation after chemotherapy.