Increasing concerns associated with the environment and global warming have triggered the exploration of the preparation of greener membranes. The abundance of renewable cellulosic biomass offers an excellent source material for membrane fabrication. In particular, cellulose acetate (CA) has been widely used as a membrane material. In this study, we explore the effect of the degree of acetylation (DoA) on membrane performance in organic solvents for the first time. We prepared CA membranes using nonsolvent-induced phase separation with Cyrene as a green solvent. Deacetylation was directly performed on the CA membranes to obtain a final DoA in the range of 1.2%–39.3%. The solvent resistance of the membranes increased as the DoA decreased. The membranes exhibited constant permeance at a pressure of 30 bar, which suggested the absence of compaction. Increasing the DoA increased the permeance of the polar aprotic solvents, whereas the permeance of the polar protic solvents showed the opposite trend. The hydrophilicity of the membranes increased with a decrease in the DoA, whereas the glass transition temperature remained quasi-constant and excellent thermal stability was maintained. The separation performance was fine-tuned with a molecular weight cut-off that ranged from 325 to 950 g mol−1 as a function of the DoA. Our results offer new avenues for tailoring solvent-resistant membrane materials from renewable polymers and solvents for application in harsh organic media.