Lipase-catalyzed acetylation of cellulose solubilized in the dimethyl sulfoxide/paraformaldehyde organic solvent system was conducted with lipase A12 from Aspergillus niger. The accompanying side cellulase activity of the A. niger lipase partly accounted for the enhanced acetylation mediated by the enzyme, via facilitating the partial degradation of cellulose substrate as evidenced by high-performance size exclusion chromatograph analysis. The enzymatic cellulose acetylation was improved by substrate pretreatment with cellulase or ultrasound by 18 and 14%, respectively, as a result of the reduced substrate molecular size. Additionally, the ultrasound-pretreated cellulose as the starting substrate was beneficial for the cellulose solution preparation due to the increased accessible surface of cellulose as evidenced by its increased sedimentation volume and SEM micrographs. The effect of thermodynamic water activity (aw) on lipase catalytic activity in organic media was also investigated. The maximum acetylation extent (nearly 11 wt %) occurred at aw = 0.52, which was improved by 51% relative to the enzymatic reaction with no control of water activity. The much larger extent to which the lipase-catalyzed cellulose acetylation was enhanced by water activity optimization than by substrate pretreatment further supported the predominant role played by the major lipase activity of the A. niger lipase over its side cellulase activity in catalyzing cellulose ester synthesis in organic media.