Butanol is the most promising alternative liquid biofuel that can substitute gasoline due to its properties which is more similar to gasoline than ethanol. In this study, butanol was produced by batch culture and repeated batch culture in acetone-butanol-ethanol fermentation (ABE) using a local strain of Clostridium acetobutylicum YM1. In batch culture using tryptone-yeast extract-acetate medium (TYA) containing 30 g/L glucose, the maximum butanol concentration was 3.78 g/L with a maximum butanol productivity of 0.08 g/L.h and the butanol yield was 0.15 g/g. Effect of some factors on the butanol production during repeated batch fermentation including drain and fill volume of medium and drain and fill time were investigated. The results of repeated batch fermentation showed that 50% (v/v) of drain and fill volume at 48 h maximized the production of butanol, productivity and yield. The highest butanol concentration was 5.12 g/L in the first cycle of repeated batch with a butanol productivity of 0.11 g/L.h and a butanol yield of 0.44 g/g. Repeated batch fermentation using free cells of Clostridium acetobutylicum YM1 eliminated the lag phase and then improved the productivity of butanol and total ABE. This study showed that repeated batch fermentation improved the efficiency of butanol production over batch culture fermentation by Clostridium acetobutylicum YM1.