The crystalline structure is a biomass recalcitrance factor that hinders chemical or biological access to degrade the plant cell-wall structure. However, controversy persists over whether a ratio of the crystalline region, the crystallinity index, is a critical biomass recalcitrance factor. In this study, an alkaline treatment modified from mercerization was adopted to alleviate the crystalline structure in the xylem of eucalyptus, along with hemicellulose and lignin removal via autohydrolysis and acid–chlorite treatment, respectively. Then, the glucose yield of the treated solid residues was used as a parameter of biomass recalcitrance. The alkaline treatment successfully reduced the crystallinity index, and the maximal reduction ratio was 84.9% when using an 8% sodium hydroxide solution. However, the reduction ratio of the crystallinity index was dependent on the remaining lignin content in the treated solid residues. Additionally, the lignin removal ratio showed critical influence to improve the glucose yield that was even observed in the treated solid residue having a low reduction ratio of the crystallinity index. Consequently, the cellulose crystalline structure is minimally involved with biomass recalcitrance, especially cellulase activity, at least in eucalyptus.