•Acorn flour has lower in vitro digestibility comparing to that of rice.•Starch hydrolase inhibitors are responsible for the low digestibility of acorn flour.•Ellagitannins are active compounds for retarding starch digestion of acorn flour.•Acorn is promising for making staple foods with low glycemic index. The acorns of Quercus fabri Hance tree, or the mandarin duck fruits have been consumed by locals in the form of noodle, yet there is little scientific study. We found that they have much lower digestibility compared with that of rice flour. Solvent extraction using hexane, dichloromethane, and acetone-ethanol-water (AEW) mixture yielded three fractions but the starch hydrolase inhibitors were only found in the AEW fraction. Using assay-guided fractionation of the extracts, we were able to further separate the active compounds by using Sephadex LH-20 column and characterize the inhibitor chemical identities by LC-MS(n). The main active compounds are ellagitannins including pedunculagin, punicalagin, castalagin, and eucalbanin. Our results indicate that the acorns from Quercus fabri Hance have potential for preparation of low glycemic index foods due to the endogenous starch hydrolase inhibitors.