Lignocellulosic biomass from corn stover holds promise as a raw material for the production of alternative energy to replace fossil fuels. In this study, structural properties of corn stover after pretreatment using mechanical pulverization with or without subsequent phosphoric acid treatment were investigated. The results showed that a pulverization step loosened the compact structure of corn stover lignocellulose and effectively reduced particle size, while both pulverization and phosphoric acid pretreatment steps altered the crystallinity index. During pretreatment, hemicellulose content was reduced and accessibility of beta-1,4 glycosidic bonds to hydrolysis by cellulase increased, while almost all lignin was retained. The results showed that the combined two-step pretreatment method improved sugar yield from lignocellulose during subsequent enzymatic hydrolysis from 20.01 mg/g to 41.41 mg/g in glucose yield. These results should guide the development of methods for improved lignocellulose conversion to sugars for enhanced bioethanol production.