The effects of sulfuric acid-associated mechanical pretreatment on the hydrolysis behavior of pine sawdust were investigated in this study. Sulfuric acid could act as an acidic catalyst to depolymerize holocellulose through cleavage of the glycosidic bonds, the dissociation energies of which were supplied by the impact of a ball on pine sawdust, during milling. The destruction of glycosidic and hydrogen bonds in pine sawdust resulted in a decrease of crystallinity and an increase of water solubility. The sulfuric acid could promote the hydrolysis of holocellulose and its hydrolysis products. It also destroyed the chemical linkages between holocellulose and lignin during ball milling. The cleavage of chemical linkages with holocellulose made lignin more difficult to hydrolyze in subcritical water, and higher activation energy was needed to hydrolyze pretreated pine sawdust at higher reaction temperatures. It also led to the formation of glucose char and aromatic-linked polymer char from the hydrolysis products of holocellulose.