As alternative microorganisms for butanol production with high butanol tolerant and productivity are in high demand, one excellent butanol-tolerant bacterium, S10, was isolated and identified as Clostridium acetobutylicum S10. In order to enhance the performance of butanol production, organic acids and neutral red were added during butanol fermentation. Synergistic effects were exhibited in the combinations of organic acids and neutral red to promote butanol production. Consequently, the optimal concentrations of combined acetate, butyrate, and neutral red were determined at sodium acetate 1.61 g/L, sodium butyrate 1.88 g/L, and neutral red 0.79 g/L, respectively, with the butanol yield of 6.09 g/L which was 20.89 % higher than that in control. These results indicated that combination of adding organic acid and neutral red is a potential effective measure to improve butanol production.