Objective：To investigate the effect of gemcitabine on hepatitis B virus（HBV） pregenomic RNA（pgRNA） transcription and HBV replication in HepG2.2.15 cells and its possible mechanisms. Methods：HepG2.2.15 cells were treated with different concentra－tions of gemcitabine，and cell viability，apoptosis，cell cycle，intracellular HBV pgRNA，and HBsAg，HBeAg，HBV DNA in the cell culture supernatant were measured by CCK8 assay，flow cytometry，qRT-PCR，and ELISA respectively. The mRNA expression levels of pgRNA transcription-related regulatory genes，hepatocyte nuclear factor 4α（HNF4α），P38 mitogen-activated protein kinase（P38MAPK），CAMP responsive element binding protein 1（CREB1），nuclear factor-kappa B p65（NF-?资b p65），histone deacetylase 1（HDAC1），per－oxisome proliferator-activated receptor γ coactivator 1α（PGC-1α），forkhead box protein O1（FOXO1），sirtuin1（SIRT1），and P53，and pgRNA in HepG2.2.15 cells treated with gemcitabine were determined by real-time quantitative PCR. The correlations between these genes and pgRNA with changes in gemcitabine concentration were analyzed to identify the gene with the highest correlation with pgRNA，and the effect of gemcitabine on the protein ex－pression of the gene was verified at the protein level using Western blot. Results：Gemcitabine showed a concentration- and dose-de－pendent inhibitory effect on HepG2.2.15 cell growth；gemcitabine increased apoptosis；gemcitabine caused more cells to arrest in G0G1 phase，and the proportion of cells in S phase decreased. Gemcitabine significantly increased HBV pgRNA in hepG2.2.15 cells in a significant time- and dose-dependent manner within 72 h；treatment of cells with gemcitabine for 24 hours increased HBeAg in the cell culture supernatant；gemcitabine promoted the mRNA and protein expression of HNF4α，and among all the genes involved in the regulation of HBV transcription we detected，the changing trend of HNF4α mRNA expression was the most correlated with that of HBV pgRNA. Conclusion：Gemcitabine can increase HBV pgRNA transcription and HBV replication at the cellular level by promoting the expression of the HBV transcription factor HNF4α.