〔Abstract〕 Objective To investigate the role of heme oxygenase-1(HO-1) on HBV replication and the antiviral effect of HO-1 combined with α-interferon(IFN-α). Methods HepG2.2.15 cells and HBV1.3-transfected HepG2 cells(HepG2-HBV1.3) were used as HBV replicating cell models; Hemin treated HepG2.2.15 and HepG2-HBV1.3 cells, to induce the expression of HO-1 molecules. CCK-8 method was used to assess the toxic effects of Hemin on HepG2 and HepG2.2.15; chemiluminescence method was used to analyze HBsAg and HBeAg in the supernatants of Hemin-treated group and si-HO-1 and other experimental groups; RT-qPCR was used to analyze HO-1, IFN-β and HBV-DNA; Western blot was used to analyze the expression of IRF-3 and the expression of related molecules in the JAK/STAT signaling pathway; Hemin combined with IFN-α treated HepG2.2.15 to monitor whether HO-1 had synergistic IFN-α antiviral effect. Results Hemin dose-dependently induced HO-1, and HO-1 was induced to exert a significant anti-HBV effect, while the expression of IFN-β, IRF-3, and IRF-9 and MxA, downstream molecules of the JAK/STAT signaling pathway, were all increased. Silencing HO-1 expression reversed the antiviral effect in the Hemin-induced group, and at the same time, type I interferon IFN-β showed low expression, and the expression of IRF-9 and MxA in the JAK/STAT signaling pathway was inhibited as well. Hemin combined with IFN-α exerted stronger antiviral effects. Conclusion HO-1 can exert an anti-HBV effect, which may be due to increased phosphorylation of IRF-3 to induce type I interferon expression and thus activate the JAK/STAT signaling pathway to exert an antiviral effect; HO-1 can synergize with IFN-α to exert an antiviral effect.