In this study, shifts in the metabolic pathways caused by pH changes during glucose catabolism by a fermentative hydrogen-producing bacterium Clostridium tyrobutyricum JM1 were determined through a metabolic engineering methodology using metabolic flux analysis (MFA). This systematic tool can provide information about the intracellular metabolic flux, which can ultimately be used for effective metabolic design or optimization, and, in this case, the maximization of hydrogen production. The objective of this work was to assess how pH affects intracellular metabolic flux and consequently the anaerobic metabolism of hydrogen-producing C. tyrobutyricum, with particular regard to hydrogen yield, which was quantified by MFA. The results of MFA indicated that the stoichiometric model could be used to adequately estimate the behavior of the system in response pH changes under anaerobic conditions.