<P><B>Highlights</B></P><P>► A novel design of co-treating <I>Populus tomentosa</I> with white rot fungi and LHW was conducted. ► Combination pretreatment performed well at hemicellulose removal and cellulose digestion than sole LHW treatment. ► A highest hemicellulose removal of 92.33% was observed by combination of <I>Lenzites betulina</I> with LHW treatment. ► Saccharification of poplar co-treated with <I>L. betulina</I> and LHW resulted in a 2.66-fold increase of glucose yield.</P> <P><B>Abstract</B></P><P>A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treatment was conducted to enhance the enzymatic hydrolysis of <I>Populus tomentosa</I>. 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 <I>Lenzites betulina</I> C5617 with LHW treatment at 200°C, which was almost 2 times higher than that of sole LHW treatment at the same level. Saccharification of poplar co-treated with <I>L. betulina</I> C5617 and LHW at 200°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 <I>Trametes ochracea</I> C6888 with LHW. The combination pretreatment performed well at accelerating the enzymatic hydrolysis of poplar wood.</P>