The inhibitory effects of 30 dietary flavonoids on dipeptidyl peptidase-IV (DPP-IV) were investigated to illustrate their quantitative structure–activity relationship (QSAR) and further explore their inhibition at the cellular level. Results of in vitro experiment show that isorhamnetin-3-O-glucoside (IC50, 6.53 ± 0.280 μM) had the strongest inhibition followed by cyanidin-3-O-glucoside (IC50, 8.26 ± 0.143 μM) and isorhamnetin-3-O-rutinoside (IC50, 8.57 ± 0.422 μM). A 3D QSAR model comparative molecular field analysis, q 2 = 0.502, optimum number of components (ONC) = 3, R 2 = 0.983, F = 404.378, standard error of estimation (SEE) = 0.070, and two descriptors; comparative similarity index analysis, q 2 = 0.580, ONC = 10, R 2 = 0.999, F = 1617.594, SEE = 0.022, and four descriptors indicates that the DPP-IV inhibition of flavonoid was facilitated by crucial structural factors. Position 3 of ring C favored bulky, hydrogen bond acceptors and hydrophilic and electron-donating substituents. The presence of minor and electron-withdrawing groups at position 4′ of ring B and positions 5 and 7 of ring A could improve DPP-IV inhibition. Moreover, the three flavonoids mentioned above could effectively suppress DPP-IV activity and expression in Caco-2 cells. This work may supply new insights into dietary flavonoids as DPP-IV inhibitors for controlling blood glucose.