 The study investigates the cooperative action of cellulase and xylonase enzymes on pretreated lignocellulosic substrates. Results show that adding xylonases to the minimum amount of cellulase required for 70% hydrolysis of steam pretreated corn stover, SPCS, or using them to partially replace the equivalent cellulase dose both result in enhanced enzymatic hydrolysis. However, the addition of xylonase increased the total protein loading required for significant improvements in hydrolysis, while partial replacement of cellulases with xylonase resulted in similar improvements without increasing enzyme loading. The enhancement resulting from xylonase-aided synergism was higher when enzymes were added simultaneously at the beginning of hydrolysis and influenced gross fiber characteristics, resulting in increased accessibility of cellulose to cellulase enzymes. This led to faster cellulose and xylan hydrolysis, a decrease in cellulase loading, and an increase in hydrolysis performance of the optimized enzyme mixture. The study also shows that the blocking effect of xylan was one of the major mechanisms limiting accessibility of cellulase enzymes to cellulose, but the synergistic interaction of xylonase and cellulase enzymes significantly improved cellulose accessibility through increasing fiber swelling and porosity. This article was authored by Sadler Jacken, Ernst Altier, and Hu Jingguang.