A novel dynamic microwave-assisted alkali pretreatment (DMAP) of cornstalk (CS) was developed in a microwave pretreatment system, which resulted in the effective removal of lignin and an increased release of soluble compounds more accessible to microorganisms. The key factors affecting the pretreatment process were optimized for enhancing thermophilic hydrogen production by co-culture of Clostridium thermocellum and Clostridium thermosaccharolyticum. The hydrogen yield reached 105.61 mL g(-1) of CS when cornstalk was pretreated by DMAP for 45 mm with an alkali loading, a liquid/solid ratio and flow rate of 0.12 NaOH g(-1) of CS, 50:1 (mL:g) and 60 mL s(-1), respectively. This was 54.8% higher than that from the untreated cornstalk, with greatly increased hemicellulose and cellulose degradations ranging from approximately 41%-79.55% and 71.28%, respectively. The present work indicates that DMAP has the strong potential to significantly improve the conversion efficiency of lignocellulosic material to renewable biofuel. (C) 2014 Elsevier Ltd. All rights reserved.