To construct expression vectors of small hairpin RNA aimed at N-acetylglucosaminyltransferase Ⅴ(GnT-Ⅴ) gene, and to investigate effects of GnT-Ⅴ shRNA on proliferation, adhesion, migration and invasion of LoVo cell line. siRNAs were designed according to the coding sequence of GnT-Ⅴ gene, shRNA expression vectors were constructed and transfected into LoVo cell line, cell lines which stably expressed low level of GnT-Ⅴwere established by G418 screening. The mRNA and protein expression of GnT-Ⅴwere measured by semi- quantitative reverse transcription polymerase chain reaction(RT-PCR) and Western blot analysis, respectively. The effects of pGPU6/GFP/Neo GnT-Ⅴ shRNA vectors on proliferation, adhesion, migration and invasion of LoVo cell line were evaluated by CCK-8 assay, heterogenous adhesion, wound closure assay, chemotactic migration and cell invasive experiment, respectively. GnT-Ⅴ shRNA expression plasmid was constructed successfully and pGPU6/GFP/Neo GnT-Ⅴ shRNA down-regulated expression of GnT-Ⅴ dramatically in LoVo cell. Expression of LoVo GnT-Ⅴ/1564 and LoVo GnT-Ⅴ/2224 dereased by 82%, 71.5% respectively at mRNA level, and 68%, 56% respectively at protein level. The more effective interfered cell line, LoVo GnT-Ⅴ/1564, was chosen to do further experiment. CCK-8 assay showed proliferation of LoVo GnT-Ⅴ/1564 was suppressed obviously, compared to proliferation of negative control group cell (P < 0.001),especially in 72 hours; down-regulation of GnT-Ⅴ expression can enhance adhesive ability(t=-3.357, P < 0.01) and inhibit chemotactic migration(t=44.051, P < 0.001) in LoVo cell line; quantitative analysis of the wound closure assay also indicated that down-regulation of GnT-Ⅴ expression can significantly prolong wound heal hours of LoVo cell line; cell invasive experiment using Matrigel gel showed that penetrative cell numbers of LoVo GnT-Ⅴ/1564 and LoVo GnT-Ⅴ/NC were 5.10±1.25 and 39.55±2.16 respectively, penetrative cell numbers of LoVo GnT-Ⅴ/1564 cell was reduced obviously, compared to negative control group cell (t=61.626, P < 0.001). The shRNA aimed at GnT-Ⅴ gene could reduce the expression of GnT-Ⅴ both in the level of mRNA and protein. By this way, it can inhibit proliferation, migration and invasion of LoVo cell line, so the sequence of RNA interference against GnT-Ⅴ may be a valid target to treat colorectal cancer.