Dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides released from foods are promising agents to combat type 2 diabetes. Millet protein supplementation can effectively ameliorate type 2 diabetes. Therefore, the objective of the study was to identify DPP-IV inhibitory peptides from millet protein and reveal the potential mechanisms. The results showed that millet protein hydrolysates (MPH) significantly inhibited DPP-IV activity compared with original millet protein (p < 0.05). MPH obtained at 3 h hydrolysis exhibited the highest DPP-IV inhibition rate (75.72 ± 1.11%). Two novel DPP-IV inhibitory peptides, Asn-Asp-Trp-His-Thr-Gly-Pro-Leu-Ser and Thr-Tyr-Pro-His-Gln-Gln-Pro-Pro-Ile-Leu-Thr, were characterized by liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. Both peptides displayed no allergenicity, hepatotoxicity and P4502D6-enzyme inhibition by in silico analysis. Further molecular docking revealed that both peptides could occupy the active center (S1, S2 subsite) of DPP-IV via H-bond and π-π. Binding energy score of Asn-Asp-Trp-His-Thr-Gly-Pro-Leu-Ser against DPP-IV was −7.270 kcal/mol, and −7.421 kcal/mol for Thr-Tyr-Pro-His-Gln-Gln-Pro-Pro-Ile-Leu-Thr. Together, these findings suggest that millet protein-derived peptides are promising in the intervention of type 2 diabetes via inactivating DPP-IV.
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