Among various routes for the biological hydrogen production, the NAD(P)H-dependent pentose phosphate (PP) pathway is the most efficient for the dark fermentation. Few studies, however, have focused on the glucose-6-phosphate 1-dehydrogenase, encoded by zwf, as a key enzyme activating the PP pathway. Although the gluconeogenic activity is essential for activating the PP pathway, it is difficult to enhance the NADPH production by regulating only this activity because the gluconeogenesis is robust and highly sensitive to concentrations of glucose and AMP inside the cell. In this study, the FBPase II (encoded by glpX), a regulation-insensitive enzyme in the gluconeogenic pathway, was activated. Physiological studies of several recombinant, ferredoxin-dependent hydrogenase system-containing Escherichia coli BL21(DE3) strains showed that overexpression of glpX alone could increase the hydrogen yield by 1.48-fold compared to a strain with the ferredoxin-dependent hydrogenase system only; the co-overexpression of glpX with zwf increased the hydrogen yield further to 2.32-fold. These results indicate that activation of the PP pathway by glpX overexpression-enhanced gluconeogenic flux is crucial for the increase of NAD(P)H-dependent hydrogen production in E. coli BL21(DE3). Biotechnol. Bioeng. 2011;108: 2941–2946. © 2011 Wiley Periodicals, Inc.