A novel aerobic denitrifying strain Methylobacterium gregans DC-1 was isolated and identified. Strain DC-1 removed 98.4% of nitrate-nitrogen (NO3--N) and 80.7% of total organic carbon with initial concentrations of 50 and 2400 mg/1, respectively. The N balance showed that most NO3--N was converted to N-2(62.18%) without nitrous oxide (N2O) emission. Response surface analysis showed that the optimal conditions for total N removal were carbon (C):N ratio of 18.7, temperature of 26.8 degrees C, pH of 6.5 and shaking speed of 180 rpm. In combination with the N balance and successful amplification of napA, nirK and nosZ genes, the metabolic pathway was as follows: NO3-NO2- -> NO -> N-2(O) -> N-2. Strain DC-1 had strong auto-aggregation rate (maximum 38.7%), produced large amounts of extracellular polymeric substances (EPS; maximum of 781.4 mg/g cell dry weight) and had corresponding strong hydrophobicity (maximum 83.2%). Pearson correlation analysis showed that EPS content and hydrophobicity were significantly positively correlated with auto-aggregation.