Abstract: A novel strain of Bacillus pumilus named as SCU11 was screened and mutagenized from the wild strain BA06 in our previous study. The fermentation supernatant of this strain showed efficient dehairing capability, indicating its extracellular proteases has a good application future in leather industry. However, the efficient genetic manipulation system of B. pumilus SCU11 has not yet been established, which greatly hampered the genetic engineering and the basic theory study of this strain. In this study, high osmolarity electroporation method was developed for the efficient transformation of B. pumilus, and the transformation efficiency was obtained up to 3.5×103CFU/μg DNA. Moreover, a temperature sensitive E. coli-Bacillus shuttle vector pUCETs was constructed by making use of a temperature sensitive replication origin, and the peptidase C40 gene of SCU11 was knocked-out based on the pUCETs. The electroporation method and gene manipulation system established in this study could make efficient targeted gene knockout in Bacliius pumilus.