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A 70% ethanol extract from azuki beans (Vigna angularis) was extracted further with CH2Cl2, EtOAc and n-BuOH to afford four fractions: CH2Cl2-soluble, EtOAc-soluble, n-BuOH-soluble and residual extract fractions. The EtOAc-soluble fractions showed the highest α-glucosidase inhibitory activity. Two pure flavonoid compounds, vitexin and isovitexin, were isolated (using the enzyme assay-guide fractionation method) from the EtOAc-soluble fractions. We further evaluated the interaction between the flavonoid compounds and α-glucosidase by fluorescence spectroscopy. Vitexin and isovitexin showed high inhibitory activities, with IC50 values of 0.4 mg·mL−1 and 4.8 mg·mL−1, respectively. This is the first study of the active compositions of azuki beans against α-glucosidase.