Clostridium tyrobutyricum is a promising organism for butyrate and n-butanol production, but cannot grow on sucrose. Three genes (scrA, scrB, and scrK) involved in the sucrose catabolic pathway, along with an aldehyde/alcohol dehydrogenase gene, were cloned from Clostridium acetobutylicum and introduced into C. tyrobutyricum (Delta ack) with acetate kinase knockout. In batch fermentation, the engineered strain Ct(Delta ack)-pscrBAK produced 14.8-18.8 g/L butanol, with a high butanol/total solvent ratio of similar to 0.94 (w/w), from sucrose and sugarcane juice. Moreover, stable high butanol production with a high butanol yield of 0.25 g/g and productivity of 0.28 g/La (TM) h was obtained in batch fermentation without using antibiotics for selection pressure, suggesting that Ct(Delta ack)-pscrBAK is genetically stable. Furthermore, sucrose utilization by Ct(Delta ack)-pscrBAK was not inhibited by glucose, which would usually cause carbon catabolite repression on solventogenic clostridia. Ct(Delta ack)-pscrBAK is thus advantageous for use in biobutanol production from sugarcane juice and other sucrose-rich feedstocks.