In order to investigate the inhibitory effect of plasmid-mediated short hairpin RNA targeting vascular endothelial growth factor (VEGF) on the expression of VEGF mRNA in human gastric cancer cells, a plasmid vector for transcribing specific short hairpin RNA targeting VEGF ( pU6-VEGF ) was constructed, andthen transfected into human gastric cancer cells using Lipofectamine2000. The VEGF mRNA expression level was detected by RT-PCR. RPMI1640 was used for blank control, and pSilencer 1.0-U6 empty plasmid for the negative control. Results showed the clone and sequence analysis revealed that the recombinant plasmid vector of pU6-VEGF was successfully constructed. The VEGF mRNA expression levels in blank control group, experimental group (pU6-VEGF) and negative control group (pSilencerl.0-U6) were 100%, 49% and 94%, respectively, indicating VEGF mRNA expression in the cells transfected with pU-VEGF vector was inhibited significantly as compared with blank control group and negative control group. It was concluded that the short hairpin RNA could significantly inhibit the expression of VEGF mRNA, which provided an experimental basis for treating human cancer with anti-angiogenesis.