The effect of a two-step cornstalk pretreating process, NaOH delignification followed by enzymolysis with cellulase and hemicellase, on dark- and photo-fermentative H-2 production was studied. A five-factor and five-level orthogonal experimental array was designed and conducted to study the effect of NaOH concentration (0-1%), hydrolysis time (0-3 h), hydrolysis temperature (98-126 degrees C), cellulase (0-18 IU/g-cornstalk) and hemicellulase dosage (0-2400 IU/g-cornstalk) on pretreatment efficiency determined by H-2 production. With NaOH 0.75%, hydrolysis temperature 108 degrees C, hydrolysis time 0.5 h, cellulase dosage 12 IU/g-CS and hemicellulase 2400 IU/g-CS, a maximum reducing sugar yield of 0.56 +/- 0.03 g/g-CS and maximum H-2 yield of 163.1 mL-H-2/g-cornstalk were obtained under dark-fermentation, and a maximum H-2 yield of 339.5 mL-H-2/g-cornstalk was obtained under photo-fermentation. According to the results, the significance of the five parameters on H-2 production was listed in high-to-low order as: NaOH concentration, cellulase dosage, hydrolysis temperature, hemicellulase and hydrolysis time. The effect of the alkaline delignification on pretreatment efficiency and the future efforts on improving H-2 production from agricultural wastes were also discussed. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.