Clostridium butyricum, a well known H2 producing bacterium, produces lactate, butyrate, acetate, ethanol, and CO2 as its main by-products from glucose. The conversion of pyruvate to lactate, butyrate and ethanol involves oxidation of NADH. It was hypothesized that the NADH could be increased if the formation of these by-products could be eliminated, resulting in enhancing H2 yield. Herein, this study aimed to establish a genetic and metabolic approach for enhancing H2 yield via redirection of metabolic pathways of a C. butyricum strain. The ethanol formation pathway was blocked by disruption of aad (encoding aldehyde-alcohol dehydrogenase) using a ClosTron plasmid. Although elimination of ethanol formation alone did not increase hydrogen production, the resulting aad-deficient mutant showed approximately 20% enhanced performance in hydrogen production with the addition of sodium acetate. This work demonstrated the possibility of improving hydrogen yield by eliminating the unfavorable by-products ethanol and lactate. Biotechnol. Bioeng. 2013; 110: 338342. (C) 2012 Wiley Periodicals, Inc.