In order to reduce the anaerobic fermentation environment and enhance the tolerance of molecular oxygen,a Clostridium acetobutylicum mutant with peroxide regulon repressors( perR) gene in-frame deletion was constructed through Group Ⅱ Intron knock-out recombination. The effect of perR knock-out on fermentation characteristics of the mutant was investigated by shake-flask fermentation experiments. The fermentation results showed that C. acetobutylicum ATCC824-δperR could enhance the yield of butanol with 7. 89% by the stationary state fermentation compared with the C. acetobutylicum ATCC 824. The yield of butanol of C. acetobutylicum ATCC 824-δperR was 3. 34 times that of C. acetobutylicum ATCC 824 when the rotate speed was 200 r / min. Results showed that C. acetobutylicum ATCC 824-δperR constructed by group II introns knock out system could reduce molecular oxygen damage in the process of fermentation,thus reduce fermentation costs.