The current standard interferon-alpha (IFN-alpha)-based therapy for chronic hepatitis C virus (HCV) infection is only effective in approximately half of the patients, prompting the need for alternative treatments. RNA interference (RNAi) represents novel approach to combat HCV by sequence-specific targeting of viral or host factors involved in infection. Monotherapy of RNAi, however, may lead to therapeutic resistance by mutational escape of the virus. Here, we proposed that combining lentiviral vector-mediated RNAi and IFN-alpha could be more effective and avoid therapeutic resistance. In this study, we found that IFN-alpha treatment did not interfere with RNAi-mediated gene silencing. RNAi and IFN-alpha act independently on HCV replication showing combined antiviral activity when used simultaneously or sequentially. Transduction of mouse hepatocytes in vivo and in vitro was not effected by IFN-alpha treatment. In conclusion, RNAi and IFN-alpha can be effectively combined without cross-interference and may represent a promising combinational strategy for the treatment of hepatitis C.