Objective: To construct recombinant lentiviral vectors expressing the shRNA against caudal-related homeobox 2 (CDX2) gene, and observe the effect of down-regulation of CDX2 gene expression on growth ability of colon cancer cells. Methods: According to CDX2 mRNA sequence, the shRNA sequences were designed and their complementary sequences were synthesized, which were then inserted into the GV248 vectors by using ligase. Positive clones were verified by DNA sequencing. Lentiviral particles were produced by cotransfection of lentiviral expression vectors with lentiviral packaging plasmids into 293T cells by lipofectamine2000, and then the viral titer was determined by serial dilution. Human colon cancer SW480 and HT29 cells were transfected with the CDX2-shRNA expressing lentiviral vectors, and then the CDX2 mRNA and protein expression were determined by qRT-PCR and Western blot analysis, and the proliferative ability was examined by CCK 8 assay and colony formation assay, respectively. Results: The results of DNA sequencing showed that CDX2-shRNA expression segments were correctly inserted into GV248 vectors, and viral titer was 1×109 TU/mL after packaging. In both SW480 and HT29 cells after transfection of CDX2-shRNA expressing lentiviral vectors, the mRNA and protein expression levels of CDX2 mRNA and protein were significantly down-regulated (all P<0.05), but no significant change was found in cell proliferation and colony forming ability (all P>0.05). Conclusion: CDX2-shRNA expressing lentiviral vectors are successfully constructed and their transfection can effectively inhibit CDX2 gene expression. Down-regulation of CDX2 gene expression exerts no obvious effect on growth of colon cancer cells.