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Starch acetate was prepared from corn starch after ultra-high pressure (UHP) pretreatment at different pressure levels (100, 200, 300, 400, 500, and 600 MPa). The structural properties of UHP treated starch and starch acetate were analyzed by scanning electron microscopy (SEM), X-ray diffraction (XRD), rapid viscosity analysis (RVA) and differential scanning calorimetry (DSC). The results indicated that both UHP treatment and acetylation reaction resulted in swelling and rupture of starch granules. After UHP pretreatment, the relative crystallinity and gelatinization enthalpy of starch increased first and then decreased, reaching maximum values of 35.4% and 11.90 J/g at 400 MPa, respectively. Meanwhile, pasting parameters reached maximum values at this pressure level. After pretreatment at 600 MPa, the starch was gelatinized completely and the morphology of starch granules was destroyed. Furthermore, the crystalline structure changed from A-type to V-type. As a result, the degree of substitution and reaction efficiency of starch acetate significantly increased, reaching maximum values of 0.103 and 81.14%, respectively, when the pressure was 500 MPa. The occurrence of the maximum values might be related to the fact that the granular state was maintained despite obvious structural damage of starch granules, resulting in increased contact between starch granules and acetic anhydride. The findings of this study demonstrate that UHP pretreatment changes the structure of starch and thus helps increase the synthesis efficiency of starch acetate.