The impact of gamma-valerolactone/water (GVL/H2O) pretreatment coupled with alkaline post-incubation on the digestibility of cellulose substrates was investigated. When Avicel was pretreated by GVL/H2O with H2SO4 as catalyst, the glucan conversion decreased from 27% (untreated Avicel) to 16% due to the esterification of cellulose after pretreatment. After incubation of GVL/H2O pretreated Avicel with 1% NaOH at 50 degrees C for 1 h, the glucan conversion enhanced to 20%. When corn stover (CS) pretreated by GVL/H2O with 25mM H2SO4 was used as substrate, alkaline post-incubation removed 72% and 50% of the hemicellulose and lignin, respectively, and increased the glucan conversion by 1.9-fold consequently. Moreover, higher cellulases adsorption capacity, affinity and binding strength on GVL/H2O pretreated CS was achieved after alkaline post-incubation. These results indicated that the improved digestibility of GVL/H2O pretreated lignocellulose by alkaline post-incubation was attributed to the depletion of ester groups on cellulose and removal of hemicellulose and lignin.