In recent years, interest in butanol as potential fuel has grown up due to its advantages over ethanol such as high energy density, phase stability on blending with other fuels, and non-corrosiveness. Clostridium acetobutylicum ATCC 824 is the most frequently used organism for butanol production by ABE fermentation. The present work was carried out to develop a strain of Clostridium acetobutylicum with improved butanol production capabilities. To achieve this objective, a gene knockout system was constructed by using an L1.LtrB group II intron from Lactococcus lactis. The transcription of the L1.LtrB intron on pCACYS3 was controlled by a thl promoter. Based on this knockout system, mutants on acetic and butyric acids pathways have been constructed from C. acetobutylicum ATCC 824 and characterized.