Cellulase, xylanase, and β-glucosidase production was studied on novel nano-shear pretreated corn stover by the mixed fungi culture. The high shear force from a modified Tayor-Couette nano-shear mixing reactor efficiently disintegrated corn stover, resulting in a homogeneous watery mash with particles in much reduced size. Scanning electron microscope study showed visible mini-pores on the fiber cell wall surface, which could improve the accessibility of the pretreated corn stover to microorganisms. Mixed fungal culture of Trichoderma reesei RUT-C30 and Aspergillus niger produced enzymes with higher cellulolytic and xylanolytic activities on corn stover pretreated with nano-shear mixing reactor, in comparison with other pretreatment methods, including acid and ammonia fiber explosion (AFEX) pretreatment. The hydrolytic potential of the whole fermentation broth from the mixed fungi was studied, and the possibility of applying the whole cell saccharification concept was also investigated to further reduce the cost of lignocellulose hydrolysis. Biotechnol. Bioeng. 2013; 110: 2123–2130. © 2013 Wiley Periodicals, Inc.