Small interfering RNA (siRNA)-induced RNA degradation can specifically inhibit viral infection and has been extensively investigated for its efficacy as an antiviral therapeutic approach. In this study we constructed a lentivirus vector carrying a U6-short hairpin RNA expression cassette to express siRNAs in vero cells. The lentivirus vector also expressed an enhanced green fluorescence protein as a reporter. Stable siRNA-expressing cell lines were successfully established, and the inhibition efficiencies of rationally designed siRNAs targeting conserved genomic regions of the Newcastle disease virus, an important disease of poultry world wide, were assessed. Our results showed that siRNAs targeting the nucleoprotein and matrix gene potently inhibited viral replication. Our study indicates that lentivirus-mediated delivery of siRNA and the resulting gene silencing can be used to study the functions of genes in viral replication and may have a potential transgenic antiviral application in poultry.