Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H 2, it can produce acids (lactic, acetic and butyric acids) and CO 2, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H 2 yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H 2 yield compared with the wild type strain. However, under low partial pressure of H 2, the hbd-deficient strain showed increased H 2 production with the simultaneous decrease of ethanol production, indicating that H 2 production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H 2 production by C. butyricum.