Objective：To establish a plasmid expressive vector coding short hairpin RNA（shRNA） of human cerb-B1 gene in vitro, and to investigate specific inhibition of cerb-B1 gene and epidermal growth factor receptor（EGFR） expression in human colon cancer LoVo cells by shRNA. Methods：DNA templa-primer of cerb-B1 gene was synthetized in vitro and the plasmid expression vectors coding shRNA were established with Pgenesil-1 plasmid. The human colon cancer LoVo cells were transfected by Lipofectamine 2000 with plasmid vector, and then selected for 4 weeks by G418. The expression of cerb-B1 mRNA was assessed by real time PCR and the protein of EGFR was assessed by Western blot method. Results：The digest identification of plasmid vectors confirmed that DNA templa-primer was successfully inserted in the plasmid, and the sequence was in conformity with the designed result. Plasmid expression vectors aiming directly at cerb-B1 gene were successfully established. After the plasmid vectors were transfected into the LoVo cells, the expression of cerb-B1 mRNA was decreased by（81.3 ± 2.8）%, and the expression of EGFR protein was decreased by（73.4 ± 2.3）%, respectively. There was a significant difference to the control plasmid vectors. Conclusion：The plasmid expression vector aiming directly at cerb-B1 gene might suppress cerb-B1 gene and EGFR expression in human colon cancer LoVo cells. The new therapeutic modalities could be used in the treatment for human colon cancer.