H 2 production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H 2 production by fermentative bacteria by maximizing the number of electrons that are available for H 2 . Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O 2 )-binding protein on oxic H 2 production in a closed batch system that was supplemented with glucose. The H 2 yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O 2 tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H 2 yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H 2 production and not by working as an O 2 -scavenger during H 2 production. This new approach allowed more substrate to remain to be further utilized for the production of more H 2 from limited resources. We expect that VHb can be successfully engineered in potential aerobic H 2 -producing microbial systems to enhance the overall H 2 production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H 2 .