In order to explore the mechanism by which ADP ribose polymerase 15 (PARP15) regulates the activation of the JAK-STAT signaling pathway by S-adenosylmethionine domain protein 2 (RSAD2) affecting the replication of hepatitis B virus (HBV). 18 HBV DNA positive patients who visited the Infection Department of Guizhou Medical University Affiliated Hospital from October 2023 to February 2024 were selected, and 18 healthy volunteers from the physical examination center were used as negative controls. Clinical data and blood samples were collected from the subjects, and an HBV infected cell model was constructed using in vitro transfection. Small interfering RNA and plasmids were transfected into the cell model, respectively. GO and KEGG enrichment were used to analyze the enrichment pathway of PARP15, while qRT PCR and Western Blot were used to detect the expression of proteins related to the PARP15 signaling pathway. ELISA was used to detect the concentrations of HBeAg and HBsAg in the cell supernatant. The results indicate that compared with the control group, the expression of PARP15 in PBMCs and serum of HBV positive patients is increased, while the expression of RSAD2 is decreases (P < 0.01). GO and KEGG enrichment analysis show that PARP15 is involved in the biological processes of the JAK-STAT signaling pathway. In the HBV infected cell model, the expression of PARP15 is higher than that of the control group (P < 0.01), while RSAD2 is lower than that of the control group (P < 0.05). Knocking down PARP15, the expression of RSAD2, STAT1, and STAT2 increase, while the expression of HBeAg, HBsAg, and HBV mRNA decrease (P <0.001). Overexpressing RSAD2, the expression of STAT1 and STAT2 also increase, while the expression of HBeAg, HBsAg, and HBV mRNA decrease (P < 0.001). Compared with cells that knocked down PARP15 alone or overexpressed RSAD2 alone, cells that knocked down or overexpressed PARP15 and RSAD2 simultaneously show a relative decrease in the expression of STAT1 and STAT2, but still higher than the control group, while the expression of HBeAg, HBsAg, and HBV mRNA is relatively upregulated but still lower than the control group (P < 0.05). It can be seen that PARP15 negatively regulates RSAD2, inhibits the JAK-STAT signaling pathway, and promotes HBV replication.