This study explored the genetic engineering of Escherichia coli for hydrogen (H2) production. In E. coli W3110, the introduction of NAD+-reducing [NiFe]-hydrogenase from Cupriavidus necator, combined with the inactivation of three endogenous [NiFe]-hydrogenases, exhibited not only H2 production but also H2 uptake based on exogenous hydrogenase. Although the H2 production ability was much lower than the H2 uptake ability, inactivation of the ethanol, lactate, and succinate production pathways resulted in a marked increase in H2 production, demonstrating the bidirectional hydrogenase function in vivo depending on NADH/NAD+. Unexpectedly, H2 production was completely repressed under conditions for high expression of exogenous hydrogenase. Furthermore, the introduction of the heterologous enzyme markedly repressed the endogenous H2 production ability of E. coli W3110 but not the HST02. These in vivo behaviors largely correlated with in vitro hydrogenase activity suggested complicated interactions between the native and nonnative functional expression of [NiFe]-hydrogenases.