A unique thermophilic fermentative hydrogen-producing strain H53214 was isolated from a deep-sea hydrothermal vent environment, and identified as Caloranaerobacter azorensis based on bacterial 16S rRNA gene analysis. The optimum culture condition for hydrogen production by the bacterium, designated C. azorensis H53214, was investigated by the response surface methodology (RSM). Eight variables including the concentration of NaCl, glucose, yeast, tryptone, FeSO4 and MgSO4, initial pH and incubation temperature were screened based on the Plackett-Burman design. The results showed that initial pH, tryptone and yeast were significant variables, which were further optimized using the steepest ascent method and Box-Behnken design. The optimal culture conditions for hydrogen production were an initial pH of 7.7, 8.3 g L-1 tryptone and 7.9 g L-1 yeast. Under these conditions, the maximum cumulative hydrogen volume, hydrogen yield and maximum H-2 production rate were 1.58 L H-2 L-1 medium, 1.46 mol H-2 mori glucose and 25.7 mmol H-2 g(-1) cell dry weight (COW) h(-1), respectively. By comparison analysis, strain H53214 was superior to the most thermophilic hydrogen producers because of the high hydrogen production rate. In addition, the isolation of C. azorensis H53214 indicated the deep-sea hydrothermal environment might be a potential source for fermentative hydrogen-producing thermophiles. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.