Objective To construct the expression vector of HLA-G-shRNA and investigate the effect of HLA-GshRNA from NK cell lysis. Methods Four HLA-G shRNA plasmids were constructed and transiently transfected to Bel-7402 cell lines， the levels of mRNA and protein of HLA-G were detected by Real-Time PCR and Western blot. The cytotoxicity of NK-92MI cells against the transfected cells was analyzed by LDH releasing assay. Results The gel electrophoresis and sequencing showed that the inserted sequence was identical to the one which we designed, and no aberrations such as mutation，deletion or insertion occurred. The expressions of HLA-G confirmed by Real Time-PCR and Western blot were significantly down-regulated. Bel-7402 cell lines transfected HLA-G shRNA showed higher lytic activity （P＜0.01）. After KIR2DL4 receptor blocked，lytic activity of NK-92 MI cell were decreased （P＜0.01）. Conclusions HLA-G shRNA plasmids are successfully constructed and HLA-G down-regulated can increase NK cytolysis against Bel-7402 cell. After HLA-G combines with KIR2DL4 receptor at the surface of NK cells， the inhibition effect is transferred.