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 nitrogen concentrations, and different kinds of carbon sources. The major hydrogen production pathway of this strain is considered to be a formate pathway. In this pathway hydrogen is produced from formate by formate hydrogen lyase (FHL). This strain produced bio-hydrogen mostly in the stationary phase, in which formate concentration was high. Optimum pH for the hydrogen production was pH 7.0, at which hydrogen production/unit volume and overall volumetric hydrogen production rate were 2615 ml/l and 154 ml H2/l/hr, respectively, at 25 g glucose/l. The maximum volumetric hydrogen production rate was estimated to be 417 ml H2/l/hr at 15 g glucose/l. Enterobacter asburiae SNU-1 was able to utilize various carbon sources such as monosaccharides, disaccharides, polysaccharide, and alcohols for the productionof hydrogen. Bacto peptone and yeast extract were favorable nitrogen sources. The concentration of nitrogen source also affected the hydrogen production.