RNA interference (RNAi) technology is a powerful tool for identifying gene functions. Chicken embryo fibroblast (CEF) is an ideal model for studying the interaction between avian viruses and their hosts. To establish a methodological platform for RNAi studies in CEF, three plasmid vectors expressing short hairpin RNAs (shRNAs) targeted against the Newcastle disease virus (NDV) NP gene were constructed. One of them, ndvl, was proven effective on blocking viral replication in CEF and chicken embryos. Four hours prior to infec-tion with NDV, the CEF was transfected with the plas-raids by Silent-fect. An unrelated shRNA sequence (HK) was used in mock transfection. The expression of a potent shRNA resulted in up to 2.3, 21.1 and 9.8 fold decreases in NP gene expression at 3, 6 and 9 h post infection in CEF, respectively. The ndvl was able to completely inhibit the replication of the virus in CEF within 48 post infection. Furthermore, the pathological changes in CEF caused by NDV were delayed, and the degree of pathological changes was lighter compared with the mock transfection in the presence of ndvl. When the complex of shRNA-Silent-fect and NDV was co-injected into the allantoic cavity of 10-day-old embryonated eggs with 105 or 106 ELD50 NDV, NDV replication was decreased by 94.14% and 62.15% after 17 h, respectively. These find-ings suggest that the newly synthesized NP protein is crit-ical for NDV transcription and replication and provide a basis for identifying the functions of viral genes and screening for effective siRNAs against viruses in CEF and chicken embryo by RNAi.