Objective：To investigate the molecular mechanism of cisplatin-induced hepatitis B virus（HBV） replication. Methods：HepAD38 cells with stable expression of HBV were treated with different concentrations of cisplatin，and the optimal concentration of cisplatin was chosen according to trypan blue staining for cell viability. The level of HBV DNA was measured by real-time PCR and Southern blot，and the expression of HBsAg and HBcAg was measured by Western blotting. The cisplatin-induced group of Hep－AD38 cells were treated with or without N-acetyl-L-cysteine（NAC）. Intracellular reactive oxygen species（ROS） levels were deter－mined with CellROX Orange Reagent under laser scanning confocal microscopy，and the level of HBV DNA and expression of HBsAg and HBcAg proteins were measured. Results：The op－timal concentration of cisplatin was 6 μmol/L according to trypan blue staining. The results of real-time PCR showed that com－pared with 515.152±18.924 in the PBS group，the copies（per cells） of HBV DNA were 1 215.758±49.001（P=0.000），1 678.182±117.223（P=0.000），and 2 110.606±143.640（P=0.000） after being treated with 0.5，2，and 6 μmol/L cisplatin，respectively. Moreover，cisplatin treatment increased HBV DNA levels in a dose-dependent manner as measured by Southern blot，the same as results of real-time PCR. The results of Western blot showed that compared with the PBS group，the 6 μmol/L cisplatin treatment group had a relative HBsAg expression level of 3.102±0.099（P=0.000） and a relative HBcAg expression level of 4.001±0.200（P=0.000），suggesting that cisplatin contributed to HBV replication and protein expression. After HepAD38 cells were treated with cisplatin and NAC，the results of real-time PCR showed that the copies（per cells） of HBV DNA in in the cisplatin group and cisplatin+NAC group were 2 180.444±82.573（P=0.000） and 1 041.778±50.918（P=0.000），respectively；there was a significant difference in the level of HBV DNA between the two groups. According to the results of Western blot，the cisplatin group had a relative HBsAg expression level of 3.139±0.061（P=0.000） and a relative HBcAg expression level of 3.812±0.109（P=0.000），and the cisplatin+NAC group had a relative HBsAg expression level of 2.101±0.088（P=0.000） and a relative HBcAg expression level of 1.613±0.073（P=0.000）；there were significant differences in the relative expression levels of HBsAg and HBcAg between the two groups. Conclusion：Cisplatin enhances HBV replication by increasing intracellular ROS levels，and NAC can partially reverse cisplatin-induced HBV replication.