Abstract: Objective: To inhibit expression of Vascular Endothelial Growth Factor (VEGF) by RNA interference and to observe the effect in different cell lines. Methods: The VEGF gene was used as the target area for RNA interference. Two specific RNA series were designed using E-RNAi services and they were put into a vector containing the U6 promoter to construct the short hairpin RNA (shRNA) expression vector. The vector was transfected into human HEK293 embryonic kidney, HT29 colon cancer, Hela cervical carcinoma and Hep G2 hepatocellular carcinoma cells using lipofectamineTM 2000. The level of VEGF mRNA was observed using RT-PCR, Northern-blot hybridization and immunofluorescent staining. Results: Two kinds of VEGF gene-specific shRNA expression vectors were successfully constructed. Effective inhibition of VEGF mRNA expression occurred in the HEK293 and HT29 cells and the inhibition ratio was 72% and 42%, respectively. However, the inhibition ratio was decreased to 28% in the HeLa cells and 13% in HepG2 cells lines. Northern blotting showed that the inhibition rate of VEGF mRNA was 88% and 89% in HEK293 and HT29 cells, respectively. Immunofluorescent staining indicated that the inhibition rate of VEGF protein expression in the HT29 cells was 73%. Conclusion: The expression vector constructed to produce shRNA targeting VEGF can effectively inhibit expression of VEGF, but there are different levels of inhibition in various cells lines.