Microalgal oil is considered a promising candidate for edible oils. However, investigation of the refining processes of microalgal oil has been limited, especially deacidification. In this work, microcrystalline cellulose (MCC) was pretreated using different methods and utilized for the first time in the deacidification of microalgal oil. Detection results from FTIR and XRD indicated alkali pretreatment had a significant effect on the structure of MCC. Some inter- and intramolecular hydrogen bonds in AMCC (alkali-pretreated MCC) were destroyed, and crystallinity index of cellulose decreased, which increased its adsorption capacity and the reaction of OH groups with free fatty acids. Some NaOH was adsorbed into AMCC through cellulose swelling, which also contributed to deacidification. The interaction with oil was also improved with many cracks and voids on the surface of AMCC. AMCC could reduce the acid value to about 2 mg KOH/g. Comparatively, original MCC and MCC pretreated with microwave or ultrasound did not exhibit the ability to deacidify. Furthermore, the conditions of alkali treatment were optimized. Treatment with 20% NaOH for 20 min was optimal. Compared with other adsorbents, such as sodium silicate and chitosan treated with alkali and resin, only AMCC could effectively reduce acid value while maintaining high lipid recovery. Therefore, AMCC was considered a better adsorbent for the deacidification of microalgal oil.