This research aimed to study the fermentative production of hydrogen using de-oiled rice bran (DRB) as renewable biomass. DRB was obtained by the extraction of oil content of rice bran samples. DRB was then hydrolyzed by dilute H2SO4 (1% v/v) to obtain DRB hydrolyzate as substrate for hydrogen generation. The composition of DRB hydrolyzate showed that glucose and xylose formed the main fermentable sugars released from acid hydrolysis of DRB. Hydrogen production was performed by the cultivation of new strain Clostridium acetobutylicum YM1 on DRB hydrolyzate in an anaerobic fermentation process. The effect of pivotal operating variables (incubation temperature, initial pH of culture medium and microbial inoculum amount) on hydrogen production was studied using response surface methodology. A second-order polynomial regression model was generated to evaluate hydrogen generation trend under conditions tested. The model analysis revealed the high significance of linear effects of incubation temperature (P < 0.01) and inoculum amount (P < 0.05) on hydrogen generation. The process model also showed that the quadratic effects of the incubation temperature and pH value were significant at 99% probability level and 95% probability level, respectively. Similar results indicated that the interaction effect between incubation temperature and inoculum amount was highly significant (P < 0.01). The regression model suggested that the optimum conditions were an incubation temperature, pH value and inoculum amount of 35.2 degrees C, 5.5 and 11.6% (v/v), respectively. In order to validate the optimum conditions determined by the model, Clostridium acetobutylicum YM1 was cultivated on DRB hydrolyzates under optimum conditions determined. Verification experiment showed that a cumulative hydrogen volume of 572.5 ml and a hydrogen yield of 132.2 ml H-2/g total sugars consumed were generated. (C) Copyright 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.