A new fermentative hydrogen-producing bacterium was isolated from a landfill, and it was identified as Enterobacter asburiae strain using a genomic DNA hybridization method. Environmental factors and metabolic flux influencing the hydrogen production were investigated, including pH, initial glucose and formate concentrations. The major hydrogen production pathway of this strain is considered to be a formate pathway by using formate hydrogen lyase (FHL). Optimum pH for the hydrogen production was pH 7.0 in PYG medium, at which hydrogen production/unit volume and overall hydrogen productivity were 2615 ml/l and 174 ml H2/l/hr, respectively, at 25 g glucose/l. The maximum hydrogen productivity was estimated to be 417 ml H2/l/hr at 15 g glucose/l. This strain produced bio-hydrogen mostly in the stationary phase, in which formate concentration was high. In this paper, hydrogen production was tried in formate medium after cell harvest.