Lignocellulose-to-hydrogen bioconversion is a promising technology to substitute fossil fuel-based energy. Adequate supplement of pretreatment to improve the digestibility of lignocellulose is required for effective fermentative hydrogen production. This study was conducted to evaluate the H-2 production from lime pretreated cornstalk and explored the mechanisms of hydrogen yield enhancement. The accumulative hydrogen yield of 155.4 ml/g-TVS was obtained at recommend conditions of lime loading 0.10 g/g biomass, ambient temperature and residence time 96 h, which was 38.1% higher than 115.3 ml/g-TVS for the untreated sample. The enhancement of the hydrogen yield was attributed to the improvement of biodegradability of the cornstalk through lime pretreatment. Chemical composition analysis showed that 23.5% lignin, 27.4% hemicellulose and 10.2% cellulose were removed, while water-soluble substances were increased by 32.3%. Microscopy observation and FTIR and XPS analysis further confirmed that the rigid structure of lignocellulose was disrupted by lime and more cellulose exposed to the surface. Crown Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.