A diet containing natural active compounds that can inhibit the hydrolytic activity of α-glucosidase on carbohydrates and intestinal glucose absorption is an effective means of controlling postprandial hyperglycemia. Phlorizin and polydatin as phenolic glycosides have a high affinity for the catalytic site of α-glucosidase, but exhibited unsatisfactory competitive inhibitory capacity, with an IC₅₀ of 0.97 and >2 mM, respectively. However, dodecyl-acylated derivatives of phlorizin and polydatin exerted α-glucosidase inhibitory capacity, with an IC₅₀ of 55.10 and 70.95 μM, respectively, which were greatly enhanced and much stronger than that of acarbose with an IC₅₀ of 2.46 mM. The SPR assay suggested the high affinity of dodecyl phlorizin and dodecyl polydatin to α-glucosidase with equilibrium dissociation constant (K_D) values of 12.0 and 7.9 μM, respectively. Both dodecyl phlorizin and dodecyl polydatin reduced the catalytic ability of α-glucosidase by reversible noncompetitive and uncompetitive mixed inhibition, which bind noncovalently to the allosteric site 2 through hydrogen bonds and hydrophobic interactions, thereby inducing the secondary structure unfolding and intrinsic fluorescence quenching of α-glucosidase. Confocal microscopy detection visually showed significant inhibitory effects on FITC-labeled glucose uptake in intestinal Caco-2 cells by phlorizin, polydatin, dodecyl phlorizin and dodecyl polydatin. In addition, based on the differentiated Caco-2 cell monolayer model, dodecyl phlorizin and dodecyl polydatin suppressed intestinal glucose transport more effectively than phlorizin and polydatin, suggesting that they were promising in vivo hypoglycemic active compounds.