Short hairpin RNA (shRNA) is an effective method for interfering gene expression and gene function research. CREB (cAMP response element binding protein), an important and conservative transcriptional factor in eukaryote, regulates many physiological functions. According to our previous research, Bombyx mori CREB (BmCREB) is closely relative to the diapause induction in the silkworm. In order to clarify the molecular mechanism of BmCREB in diapause induction, we constructed the interfering vector pMD18-U6-shRNA of BmCREB shRNA and transfected in a Bombyx mori ovary cell line (BmN). The expression of BmCREB was detected by Western blot and RT-PCR. The results showed that BmCREB was efficiently reduced by 63.0% with the transfection of 1 μg T-227 plasmid in 72 hours. The down-regulation of BmCREB was significant within different shRNA plasmids. Plasmid T-227 had the best efficiency. These results would lay a foundation for the further research on BmCREB mechanism of the Bombyx mori diapause by shRNA in vivo.