This study investigates the improvement of butanol production in the presence - Tween 80 by Clostridium acetobutylicum NJ4. With supplementation of 15% Tween 80 (v/v), the final butanol titer could be significantly enhanced to 18.65 g/L from 70 g/L glucose, which is 38% higher than that in the control batch (13.47 g/L). With further in situ extraction using biodiesel, the butanol titer was finally improved to 33.90 g/L in the fed batch mode. The supplementation of Tween 80 could improve butanol tolerance of strain NJ4 up to 18 g/L compared to 10 g/L of control. Meanwhile, butanol dehydrogenase activity was improved to 3.89 U/mg, which is 80.93% higher than that of the control. These factors may attribute to the high butanol production. The finding of this study thus offers fundamental knowledge for the future development of alternative ABE production strategies.