This work investigates the effect of different anti-solvents (water, ethanol, or both water and ethanol) on the characteristics of cellulose dissolved and then generated from 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). Compared with original microcrystalline cellulose (MCC) granules, all regenerated celluloses showed a homogeneous, agglomerated macromorphology and had its crystalline structure transformed from original cellulose I to cellulose II. The regenerated cellulose using water (43.3%) had a higher degree of crystallinity than that using ethanol (13.5%), and a degree of crystallinity of 21.3% was obtained when an ethanol-water-ethanol treatment method was used. SAXS and FTIR results indicate that water as an anti-solvent could promote the rearrangement of cellulose molecular chains and the rebuilding of an ordered aggregated structure. Moreover, the regenerated cellulose with water showed better thermal stability than that of the samples regenerated using ethanol. Thus, our results suggest that the reconstitution of cellulose molecules during regeneration with various anti-solvents can affect the multiscale structures and properties of cellulose.