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<SUB>2</SUB> 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.