Bio-butanol as a potential alternate liquid fuel for fossil fuel overcomes many problems associated with conventional alternate liquid fuels such as ethanol and biodiesel.However, existing fermentations are limited in low butanol productivity.One excellent butanol-tolerant bacterium, S 10, was isolated from the medium supplement with high level of butanol in this study.Strain S10 was identified and designated as Clostridium acetobutylicumS10, based on the 16S rDNA analysis.Organic acids and neutral red were added to enhance the performance of butanol production.Results show the best time for adding regulators is the end of bacterial logarithmic phase.Optimal concentrations of metabolic regulators were determined by using Box-Behnken experiment and the butanol yield and butanol percentage of total solvent increased by 20.89% and 18.35%, respectively.Results demonstrated metabolic regulation was effective and strain S10 might be an alternative candidate for bio-butanol production.