A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa. The results showed that lignin and cellulose increased with the elevating temperature, while significant amount of hemicellulose was degraded during the LHW pretreatment. A highest hemicellulose removal of 92.33% was observed by combination of Lenzites betulina C5617 with LHW treatment at 200 degrees C, which was almost 2 times higher than that of sole LHW treatment at the same level. Saccharification of poplar co-treated with L. betulina C5617 and LHW at 200 degrees C resulted in a 2.66-fold increase of glucose yield than that of sole LHW treatment, and an increase (2.25-fold) of glucose yield was obtained by the combination of Trameres ochracea C6888 with LHW. The combination pretreatment performed well at accelerating the enzymatic hydrolysis of poplar wood. (C) 2011 Elsevier Ltd. All rights reserved.