The glucose-mediated carbon catabolite repression (CCR) in Clostridium tyrobutyricum impedes efficient utilization of xylose present in lignocellulosic biomass hydrolysates. In order to relieve the CCR and enhance xylose utilization, three genes (xylT, xylA, and xylB) encoding a xylose proton-symporter, a xylose isomerase and a xylulokinase, respectively, from Clostridium acetobutylicum ATCC 824 were co-overexpressed with aldehyde/alcohol dehydrogenase (adhE2) in C. tyrobutyricum (ack). Compared to the strain Ct(ack)-pM2 expressing only adhE2, the mutant Ct(ack)-pTBA had a higher xylose uptake rate and was able to simultaneously consume glucose and xylose at comparable rates for butanol production. Ct(ack)-pTBA produced more butanol (12.0 vs. 3.2g/L) with a higher butanol yield (0.12 vs. 0.07g/g) and productivity (0.17 vs. 0.07g/Lh) from both glucose and xylose, while Ct(ack)-pM2 consumed little xylose in the fermentation. The results confirmed that the CCR in C. tyrobutyricum could be overcome through overexpressing xylT, xylA, and xylB. The mutant was also able to co-utilize glucose and xylose present in soybean hull hydrolysate (SHH) for butanol production, achieving a high butanol titer of 15.7g/L, butanol yield of 0.24g/g, and productivity of 0.29g/Lh. This study demonstrated the potential application of Ct(ack)-pTBA for industrial biobutanol production from lignocellulosic biomass. Biotechnol. Bioeng. 2015;112: 2134-2141. (c) 2015 Wiley Periodicals, Inc.