Interferon alpha (IFN-alpha) is commonly used for the treatment of chronic hepatitis B (CHB) patients. Many factors including viral genetics may determine the outcome of IFN-alpha therapy. In this study, we tested whether the expression of IFN-alpha directly in the liver inhibits HBV gene expression and replication using a HBV hydrodynamic injection (HI) mouse model. Two replication-competent clones from different HBV isolates that belonging to HBV genotype A and B based on a pAAV vector (pAAV-HBV-A and pAAV-HBV-B) were compared for their susceptibility to IFN-alpha. HBV clones were injected into mice either alone or in combination with a murine (m) IFN-alpha expression plasmid (pmIFN-alpha). HBsAg and HBeAg concentrations and HBV DNA levels in mice differed after injection of these two HBV clones. Co-application of pmIFN-alpha together with the two distinct isolates resulted in markedly different kinetics of decline of HBsAg, HBeAg, and HBV DNA levels in the mice. Immunohistochemical staining of liver sections with anti-HBc showed that mIFN-alpha application completely inhibited the expression of HBcAg in mice inoculated with pAAV-HBV-B, whereas the expression of HBcAg was only reduced in mice with pAAV-HBV-A. Consistently, mice injected with pAAV-HBV-B and pmIFN-alpha showed higher expression levels of the IFN-stimulated genes (ISGs) ISG15, OAS, PKR as well as proinflammatory cytokine IL-6 in the liver. In addition, expression levels of anti-inflammatory cytokine IL-10 was down-regulated significantly in liver of the mice injected with pAAV-HBV-B and pmIFN-alpha. Our data demonstrate that IFN-alpha exerts antiviral activity in HBV mouse model, but different HBV isolates may have diverse susceptibility to IFN-alpha.