AbstractAlkali pretreatment is one of the leading pretreatment technologies for biofuel applications. The histochemical and structural characteristics of poplar cell walls were investigated before and after sodium hydroxide pretreatment (121 oC, 2%) to understand the alterations in biomass cellular structure, which were correlated with saccharification yield. Results showed that alkali pretreatment preferentially removed lignin from the S2 of fibers, which was similar to the behaviors of coniferyl alcohol and aldehyde (lignin-CAA), exhibiting a positive correlation between removal of the two structures. Additionally, the cellulose microfibril angle was enlarged as the residence time increased during pretreatment. Scanning electron microscopy (SEM) analysis further suggested that pretreatment caused ultrastructure changes in cell walls with cracks formation on cell wall surface, especially in the areas adjacent to the cell corner middle lamellar (CCML). Accordingly, the cellulose digestibility of residues increased from 32.1% for the raw material to 53.7% for the treated samples obtained in 72 h. It can be concluded that the changes in topochemistry and ultrastructure of poplar cell walls resulting from alkali pretreatment mediated the efficiency of enzymatic hydrolysis of residues.