This study reports a unique acetone uncoupled Clostridium species strain CT7, which shows efficient capability of glycerol utilization with high butanol ratio. Medium compositions, such as substrate concentration, micronutrients and pH show significant effects on butanol production from glycerol by strain CT7. To further maximize butanol production, fermentation conditions were optimized by using response surface methodology (RSM). Final butanol production of 16.6 g/L with yield of 0.43 g/g consumed glycerol was obtained, representing the highest butanol production and yield from glycerol in the batch fermentation mode. Furthermore, strain CT7 could directly convert crude glycerol to 11.8 g/L of butanol without any pretreatment. Hence, strain CT7 shows immense potential for biofuels production using waste glycerol as cheap substrate.