Abstract: Inhibiting α-glucosidase activity effectively controls the postprandial fluctuations in blood glucose derived from starchy foods. To investigate the inhibitory effects of genistein on α-glucosidase activity, we performed in vitro digestion assays to determine the effects of genistein on starch digestion characteristics. The type and molecular mechanism of inhibition were examined based on enzyme kinetics, fluorescence spectroscopy, and molecular docking analyses. The results indicated that treatment with genistein significantly reduced the content of rapidly digestible starch (P<0.05) and promoted notable increases in the content of slowly digestible starch and resistant starch (P<0.05). Genistein inhibited α-glucosidase dose dependently, with an IC₅₀ value of 1.10±0.06 mg/mL, and the inhibition type was identified to be predominantly competitive within a mixed reversible inhibition. Genistein quenching the intrinsic fluorescence of α-glucosidase with a mixed pattern by binding to the ground state fluorescent groups and colliding with the excited state fluorescent groups. The binding free energy from molecular docking was less than -12 kJ/mol, indicating that the binding of genistein to α-glucosidase could occur spontaneously. Moreover, we found that genistein targeted the hydrophobic pocket of α-glucosidase, forming hydrophobic interactions with residues Glu-429, Lys-432, and Ile-419, and hydrogen bonds with Gly-161, Asn-235, Asn-317, Glu-422, and His-423. These interactions contributed to stabilizing the binding of genistein to α-glucosidase, thereby reducing its catalytic activity. Our findings provide theoretical support for the application of genistein in functional foods for blood glucose stabilization.