Trypsin-treated β-lactoglobulin significantly decreased the glucose level after an oral glucose tolerance test using mice. We performed the present study to identify the active peptide inhibiting dipeptidyl peptidase-4 from trypsin-treated β-lactoglobulin. Trypsin-treated β-lactoglobulin showed a concentration-dependent inhibition for dipeptidyl peptidase-4, with an IC(50) value of 210 µM, although non-treated β-lactoglobulin showed no significant effect in the in vitro assay. The active peptide was isolated from trypsin-treated β-lactoglobulin and identified as the hexapeptide Val-Ala-Gly-Thr-Trp-Tyr (β-lactoglobulin f15-20). This hexapeptide also exhibited a concentration-dependent inhibitory effect and IC(50) value was 174 µM, suggesting that this hexapeptide is almost totally responsible for the DPP-4 inhibitory activity of trypsin-treated β-lactoglobulin.