The effect of D-glucose on the anaerobic production of hydrogen (H2) in the dark by bidirectional hydrogenase of Synechocystis sp. strain PCC 6803 has been studied. D-glucose addition enhanced H2 production rate. It is deduced that NAD(P)H, which is a substrate of H2 production reaction, was supplied by catabolism of D-glucose. The molar consumption ratio of H2 and total sugar consumption was low in the presence of D-glucose due to production of L-lactic acid and other metabolites. The cells photosynthetically grown in air produced lower H2 in the dark as compared with those grown in air with 6% CO2, but the lower H2 production was compensated with the addition of D-glucose at 6.4 times of control value without D-glucose. The trend of effect of pH in a growth medium and a reaction mixture on H2 production was not affected by the presence of D-glucose. This result implies that mechanisms of H2 productions with and without D-glucose are similar.