Japanese encephalitis virus (JEV), a serious mosquitoborne flavivirus, causes an acute infection of the central system resulting in encephalitis of humans and many kinds of animals. A high proportion of the survivors exhibit neurogical and psychiatric sequelae. NS1 is one of important non-structural proteins, which was found to be associated with viral RNA replication. To inhibit NS1 expression, four small interfering RNAs (siRNAs) expression plasmids (pS-NS1A, pS-NS1B, pS-NS1C and pS-NS1D) were generated to target four different coding regions of the NS1 gene, and were separately co-transfected into Vero cells with an NS1-EGFP fusion expression plasmid pNS1-EGFP. NS1 expression was evaluated by fluorescence microscope, flow cytometry assay, Western blot and RT-PCR. The results revealed that pS-NS1B, pS-NS1C and pS-NS1D could effectively and specifically inhibit NS1 expression in Vero cells. Our data suggested that these siRNAs could be used to inhibit JEV replication by silencing NS1 protein expression in further study.