Cotton fibers mercerized under the relaxed state were hydrolyzed with crude cellulase. The mercerization treatment examined included ammonia treatment, sodium hydroxide treatment, and two combined treatments using ammonia and sodium hydroxide. Crystalline regions of the mercerized fibers were hydrolyzed in the first step of hydrolysis. In this step, ammonia treatment decreased the crystallite size to a great extent due to the hydrolysis of the cellulose III crystalline phase. Cellulase treatment rendered the crystallite surface highly accessible to water molecules. The crystalline phase was closely related to water sorption of cellulase-treated fibers. The sequence of treatment had an influence on the fiber structure in the case of the combined mercerization treatment with ammonia and sodium hydroxide.