The anaerobic H2 production in the dark by bidirectional hydrogenase of unicellular cyanobacterium Synechocystis sp. strain PCC 6803 has been studied. D-glucose addition to cell suspension has been known to enhance H2 production, and the two mutants, glucose tolerant strain and its mutant lacking L-lactate dehydrogenase (LDH) gene, were introduced to improve H2 production with D-glucose. Higher D-glucose uptake rate of glucose tolerant strain resulted in higher H2 production rate comparing with the wild type strain. The LDH mutant showed higher initial H2 production rate and H2 yield from D-glucose than the other strains because the NADH which should be consumed originally by LDH can be redistributed to the H2 production reaction.