Objective:To study the inhibitory effect of ezetimibe in an experimental model of human hepatoma cell line (HepaRG) infected with hepatitis B virus (HBV) positive human serum in vitro.Methods:Mature HepaRG cells were divided into a treatment group (received drugs) and a control group (did not receive drugs). In the ezetimibe prevention experiment, the cells in the treatment group was treated with drugs 2 h before infection and 24 h during infection. In the ezetimibe treatment experiment, the cells in the treatment group were treated with drugs for 6 ~ 10 days continuously after 24 hours of HBV infection. The expression of HBV DNA and intracellular cccDNA in the supernatant was detected by fluorescence quantitative PCR. Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) content in the cell supernatant were detected by chemiluminescence. Analysis of variance was used to compare the differences between multiple groups. Pairwise comparisons among groups were followed by t- test with normal distribution. P < 0.05 was considered as statistically significant. Results:Ezetimibe prevention experiment showed that compared with control group, the treatment group was added with 20, 60, and 100 μmol/L ezetimibe before and during infection, and the HBV DNA content in the supernatant 2 days before was significantly reduced ( P < 0.05) in the treatment group. Compared with the control group, the HBsAg expression level 2 days before was significantly reduced ( P < 0.05) with the addition of 60 μmol/L ezetimibe in the treatment group. Compared with the control group, the expression level of intracellular cccDNA was significantly reduced ( P < 0.05) after 10 days with the addition of 100μmol/L ezetimibe in the treatment group. Ezetimibe treatment experiment showed that cccDNA content in the cells were significantly lowered with the immediate addition of 60μmol/L ezetimibe 24 hours after infection for 10 days when compared to control group ( P < 0.05). Conclusion:Ezetimibe, as a cytosolic inhibitor, has a certain inhibitory effect on hepatitis B virus infection in both prevention and treatment experiment.