In the present study, we identified an effective siRNA targeting M2 gene (siM2), a highly conserved gene in IAV, as compared to a reported effective siRNA targeting NP gene (siNP). We further established cell lines which stably expressing the shRNAs by transducing lentiviral-shRNA vectors to Madin-Darby cannie kidney (MDCK) cells. Using these two cell lines, we evaluated long term antiviral effects of these siRNAs against IAV subtypes H1N1 and H5N1 and further screened the potential siRNA-resistant viral mutations. Our results showed that rationally designed siM2 conferred long term effective inhibition for IAV replication. It was further demonstrated that no siRNA-resistant viral mutation appeared in siM2 targeting sequence even after the virus was cultured in the shRNA expressing stable cell line for 40 passages.