Sugarcane bagasse (SCB) used in hydrogen production by Clostridium butyricum was hydrolyzed using H2SO4 at various concentrations (0.25-7.0% volume) and reaction times (15-240 min) at 121 degrees C, 1.5 kg/cm(2) in autoclave. Optimal conditions obtained were 0.5% H2SO4 and 60 min which yielded 24.5 g-COD/L of total sugar. At these conditions, 11 g glucose/L; 11.29 g xylose/L; 2.22 g arabinose/L; 2.48 g acetic acid/L and 0.12 g/L furfural were obtained. Effects of initial pH and substrate concentration on the bio-hydrogen production from SCB hemicellulose hydrolysate by C. butyricum were then investigated. The best hydrogen yield of 1.73 mol H-2/mol total sugar and the hydrogen production rate of 1611 mL H-2/L/day were obtained at the initial pH 5.5 and initial sugar concentration 20 g-COD/L and compared very favorably with those reported in literature. Results suggested the possibility of using SCB hemicellulose hydrolysate as a fermentation media for hydrogen production by C. butyricum. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.