To enhance butanol tolerance and prevent productive degeneration in Clostridium acetobutylicum during long-term preservation, a novel approach called 1-butanol-glycerol storage was developed in this study. The viability, butanol tolerance and productivity of cells were investigated under each condition of storage within 12 months, respectively. The results suggested that the optimal condition of storage was in the solution containing 16 g L-1 butanol coupled with 200 g L-1 glycerol at 37 degrees C not -20 degrees C or 4 degrees C. Under this condition, C. acetobutylicum D64 primarily obtained the cell survival rate of 80% after 12 months. Unexpectedly, these cells still got an enhanced butanol tolerance of 32 g L-1 which was similar to 1 fold higher than the wild-type with butanol tolerance of 16 g L-1. Moreover, the butanol production has also got a little of increase but not dropped compared to the control. These results indicated this culture preservation method be available for enhancing butanol tolerance and preventing productive degeneration of C. acetobutylicum. (C) 2014 Published by Elsevier Ltd.