Abstract Background Pretreatment is an essential step in the enzymatic hydrolysis of biomass for bio-ethanol production. The dominant concern in this step is how to decrease the high cost of pretreatment while achieving a high sugar yield. Fungal pretreatment of biomass was previously reported to be effective, with the advantage of having a low energy requirement and requiring no application of additional chemicals. In this work, Gloeophyllum trabeum KU-41 was chosen for corn stover pretreatment through screening with 40 strains of wood-rot fungi. The objective of the current work is to find out which characteristics of corn stover pretreated with G. trabeum KU-41 determine the pretreatment method to be successful and worthwhile to apply. This will be done by determining the lignin content, structural carbohydrate, cellulose crystallinity, initial adsorption capacity of cellulase and specific surface area of pretreated corn stover. Results The content of xylan in pretreated corn stover was decreased by 43% in comparison to the untreated corn stover. The initial cellulase adsorption capacity and the specific surface area of corn stover pretreated with G. trabeum were increased by 7.0- and 2.5-fold, respectively. Also there was little increase in the cellulose crystallinity of pretreated corn stover. Conclusion G. trabeum has an efficient degradation system, and the results indicated that the conversion of cellulose to glucose increases as the accessibility of cellulose increases due to the partial removal of xylan and the structure breakage of the cell wall. This pretreatment method can be further explored as an alternative to the thermochemical pretreatment method.