Clostridium tyrobutyricum, a strict anaerobic, gram-positive bacterium, produces acetate, butyrate, and hydrogen from different saccharide including glucose and xylose during anaerobic fermentation. According to the metabolic pathway, 4 mol of hydrogen could be produced when 1 mol of glucose is converted to acetate while 2 mol of hydrogen could be produced if butyrate is the only co-product. In order to increase the hydrogen production, a ptb-deleted mutant has been constructed by the homologous recombination to decrease the butyrate production. The ratio of butyrate/acetate decreased 20% and the hydrogen production also increased in the mutant. Fermentation conditions such as pH and substrate initial concentration are also optimized to continually increase the hydrogen production.