Biohydrogen production from corncob by dark fermentation was reported for the first time. The effects of the pretreatment condition, substrate concentration and initial pH on the hydrogen production were investigated in batch cultivations. The maximum hydrogen yield of 107.9 mL/g-TVS and hydrogen production rate of 4.2 mL/g-TVS .h(-) were obtained under the condition of 1% HCl pretreating substrate for 30 min, 10 g/L substrate concentration and initial pH8.0. The content of hemicellulose in corncob decreased significantly from 42.2% to 3.0% after HC1 pretreatment. The contents of cellulose, hemicellulose and lignin in the acid pretreated corncob decreased slightly in hydrogen producing process. The results indicate that the acid pretreatment of the substrate plays a key role in the conversion of corncob into biohydrogen. Fourier transform infrared spectroscopy (FTIR) was used to study the changes in the corncob composition during the treatment of chemical-microbial process. It was shown that the amorphous domains of cellulose and hemicellulose were hydrolyzed into fermentable asccharides through HCl pretreatment and the microorganisms had a devastating effect on the crystallinitiy of the cellulose.