The feasibility of utilizing corn straws to produce butanol via fermentation with Clostridium acetobutylicum was evaluated. The supernatant of enzymatically hydrolyzed supernatant of steam-exploded corn straws was used as the raw material. A bacterial strain was selected from Clostridium acetobutylicum zzu-02 and Clostridium beijerinckii zzu-01, which was capable of fermenting the enzymatically hydrolyzed supernatant of steam-exploded corn straw to produce butanol with high yield. The optimal fermentation conditions for the selected strain with enzymatically hydrolyzed supernatant of steam-exploded corn straw were also investigated and they were determined as follows: sugar concentrations in enzymatically hydrolyzed solution of steam exploded corn straws, 57.5 g/L; initial pH, 6.3; the amount of added CaCO3, 5g/L; the bacterial inoculation concentration to enzymatically hydrolyzed solution, 6%; fermentation temperature, 37 oC, the amounts of the added nutritional elements, i.e. yeast extract, CH3COONH4, KH2PO4, and C6H6N2O, 0.8, 6.0, 0.5, and 0.25 g/L, respectively. Under these conditions, the butanol yield reached 9.88 g/L. Based on the butanol metabolism pathways, supplementation of a small amount of C6H6N2O was found to effectively increase the yield of butanol production.