The biohydrogen production from rice straw hydrolyzate in a continuously external circulating bioreactor (CECBR) was carried out in this study. The rice straw hydrolyzate was obtained by a concentrated sulphuric acid pretreatment. The original hydrolyzate concentration was 40-50 g total sugar/L. The feeding concentration of hydrolyzate was adjusted to 20 g total sugar/L by tap water. The working volume of CECBR was 300 mL with a height of 22.5 and a width of 7.5 cm respectively. The positions of external circulating ports were 13 and 5 cm high of CECBR with volumetric circulating rate of 9.6 L/min. The average hydrogen production rates (HPR) of 5.52 L/L/d (hydrogen molar yield: 0.72 mol H-2/mol hexose) and 16.32 L/L/d (hydrogen molar yield: 1.02 mol H-2/mol hexose) were obtained at hydraulic retention time (HRT) 8 and 4h respectively. The value of hydrogen production rate at HRT 4 h was three times more than that at HRT 8 h. The presence of bacteria Clostridium favors the hydrogen production from the acetic acid and butyric acid metabolic pathways which were determined by DGGE analysis. The biomass could not be maintained in the CECBR by washing out of the bacteria when the feeding rate was lowered to HRT 2 h. This phenomenon resulted in less hydrogen production and ineffective degradation of hydrolyzate. But the continuously external circulating bioreactor could be steady operated at HRT 4 h with an impressive hydrogen production rate by a rice straw hydrolyzate. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.