HCV and HBV are the major causes of chronic liver diseases worldwide. Patients with both viruse's co-infection tend to develop severer liver diseases and are at high risk of liver-related death. NS5A protein of HCV plays key roles in HCV replication and inhibition of host immune responses. In this study, we described the establishment of HepG2-derived cell line that stably expresses NS5A protein and the application of a cellular system for HBV replication based on a recombinant adenovirus carrying HBV genome. Our results demonstrated that NS5A enhances the expression of S and E proteins of HBV, as well as the synthesis of viral DNA. Moreover, we showed that NS5A assists HBV to escape interferon responses. These data suggested that NS5A of HCV may employ multiple strategies contributing to the enhancement of HBV replication and interferon resistance during the co-infection of HCV and HBV.