Clostridium tyrobutyricum does not have the enzymes needed for using maltose or starch. Two extracellular alpha-glucosidases encoded by agluI and agluII from Clostridium acetobutylicum ATCC 824 catalyzing the hydrolysis of alpha-1,4-glycosidic bonds in maltose and starch from the non-reducing end were cloned and expressed in C. tyrobutyricum (Delta ack, adhE2), and their effects on n-butanol production from maltose and soluble starch in batch fermentations were studied. Compared to the parental strain grown on glucose, mutants expressing agluI showed robust activity in breaking down maltose and produced more butanol (17.2 vs. 9.5 g/L) with a higher butanol yield (0.20 vs. 0.10 g/g) and productivity (0.29 vs. 0.16 g/L h). The mutant was also able to use soluble starch as substrate, although at a slower rate compared to maltose. Compared to C. acetobutylicum ATCC 824, the mutant produced more butanol from maltose (17.2 vs. 11.2 g/L) and soluble starch (16.2 vs. 8.8 g/L) in batch fermentations. The mutant was stable in batch fermentation without adding antibiotics, achieving a high butanol productivity of 0.40 g/L h. This mutant strain thus can be used in industrial production of n-butanol from maltose and soluble starch.