To improve butanol tolerance and production in Clostridium acetobutylicum, a novel approach was developed in this study, which was called artificial simulation of bio-evolution (ASBE) based on the evolutionary dynamics and natural selection. Through repetitive evolutionary domestications, a butanol-tolerant strain C. acetobutylicum T64 was obtained, which could withstand 4% (v/v) (compared to 2% of the wildtype) butanol and was accompanied by the increase of butanol production from 12.2 g/L to 15.3 g/L using corn meal as substrate. Fermentation was also carried out to investigate the relationship between butanol tolerance and ABE production, suggesting that enhancing butanol tolerance could increase butanol production but unlikely improve total ABE production. These results also indicated that the ASBE would be an available and feasible method used in biotechnology for enhancement of butanol tolerance and production. (C) 2012 Elsevier Ltd. All rights reserved.