Objective:To construct the full-length nonstructal-1 (NS1) gene of influenza A (H3N2) into a eukaryotic expression vector and express NS1 gene in mammalian cells,as well as to investigate the effects of NS1 on proliferation of 293T cells. Methods:The NS1 gene of influenza A (H3N2) was amplified by RT-PCR and cloned into pMD18-T simple vector to construct a plasmid,named pMD18-T/NS1. the pMD18-T/NS1 and the pXJ40-HA were double-digested,to yield the recombinant eukaryotic expression vector pXJ40-HA-NS1. The expression of the NS1 gene in transfected 293T cells was identified by Western blot. The effects of NS1 gene on cell proliferation were measured with 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide(MTT) method. Results: The recombinant eukaryotic expression vector pXJ40-HA-NS1 was successfully constructed. The expression of NS1 protein was detected by Western blot. NS1 over-expression inhibited the proliferation of 293T cells. Conclusion: The full-length NS1 gene has been obtained and its recombinant eukaryotic expression vector has been successfully constructed. And we found that the proliferation was dramatically inhibited in 293T cells over-expressed with NS1 protein. The construction of eukaryotic expression plasmid of NS1 gene made it possible to set up the cell model expressing NS1 protein and further study the effects of NS1 protein on cell proliferation and apoptosis.