Objective：To construct a lentiviral vector for RNA interference（RNAi） of Rabex-5 gene and to detect its silence effect on Rabex-5 in MCF-7 cells. Methods：The synthesised single-stranded primer was annealed to double-stranded oligo sequences and sub-cloned into linear pMAGic lentiviral plasmid vector digested by enzyme AgeⅠ and EcoRⅠ. DH5?琢 competent cells were trans－formed and positive clone were screened，identified and sequenced. The recombinant lentivirus was packaged into mature lentivirus by 293FT cells and used to infect MCF-7 cells. The expression of Rabex-5 in the cells was detected by real-time PCR and West－ern blot. Results：PCR and sequencing verified that the recombinant lentivirus plasmid Rabex5-shRNA was successfully constructed. Compared with the negative control and the blank control cells，Rabex-5 mRNA and protein level was reduced in MCF-7 cells in－fected by lentivirus. RNAi3 had the best effect of gene silence. Conclusion：The lentiviral RNAi expression vector targeting Rabex-5 gene is successfully constructed and it can downregulate the expression of Rabex-5 in MCF-7 cells，which lays the experimental foundation for the research on the changes of malignant biological activity of tumor cell lines and gene therapy.