As a dimer of proanthocyanidin, proanthocyanidin B2 (PB2) was found to effectively attenuate postprandial blood glucose in mice after sucrose loading. Further studies showed that PB2 revealed strong inhibitory activity against α-glucosidase with an IC50 value of (0.23 ± 0.01) μg mL-1, and this process was reversible with a mixed-type inhibitory manner. PB2 quenched the intrinsic fluorescence of α-glucosidase through a static quenching mechanism and changed the micro-environments and conformations of α-glucosidase that led to an aggregation phenomenon. A further molecular docking study provided more information about the interactions of PB2 with the amino acid residues of α-glucosidase. Our results suggest that PB2 is useful for protection against hyperglycemia through inhibiting the activity of α-glucosidase.