Dipeptidyl peptidase-IV (DPP-IV) is an enzyme that break down the antidiabetic hormone glucagon-like peptide-1. Therefore, inhibition of DPP-IV could be an effective strategy to treat Type 2 diabetes (T2D). The α-lactalbumin-rich whey protein concentrate was hydrolyzed by trypsin, and the hydrolysates were then fractionated at a semi-preparative scale using a Superdex Gel filtration Chromatography. The peptides were analyzed by using HPLC coupled with tandem mass spectrometry (RP-HPLC-MS/MS), and their Dipeptidyl peptidase-IV inhibitory activity was determined by the enzymatic assay. Among tested fragments, a potent fragment (LDQWLCEKL), with the half-maximal inhibitory concentration (IC50) of 131 μM was obtained. Further analysis shows that the LDQWLCEKL peptide corresponds to the amino acid sequence of f(115-123) in α-lactalbumin. Furthermore, LDQWLCEKL exhibited a typical non-competitive mode of inhibition. The results indicate that α-lactalbumin contains active peptides with DPP-IV inhibitory activity that may be used to prevent and treat T2D.