β-Lactoglobulin (BLG) is the most abundant protein in cow’s milk whey. Peptides prepared from BLG digested with the gastrointestinal (GI) proteases pepsin and trypsin strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity. DPP-IV is a ubiquitously expressed serine protease that cleaves N-terminal dipeptides from incretins, which are insulinotropic GI hormones. Recently, genetically modified lactic acid bacteria have emerged as efficient cell factories for the production and safe delivery of heterologous proteins to the GI tract. Here, we engineered a strain of Lactococcus lactis to secrete recombinant BLG (rBLG) and evaluated the DPP-IV-inhibiting activity of trypsin-digested rBLG. To construct rBLG-producing L. lactis, a codon-optimized bovine BLG gene was inserted into the lactococcal secretion vector pNZ8148#2:SEC, containing a nisin-inducible promoter and signal peptide sequence from USP45 protein. The constructed rBLG secretion vector was transformed into L. lactis NZ9000 (NZ9000) as a host strain for gene expression. The secretion of rBLG by NZ9000 was confirmed by Western blotting. The DPP-IV-inhibitory activity of trypsin-digested rBLG was examined by comparative analysis with trypsin-digested commercial BLG (cBLG) using a DPP-IV Drug Discovery Kit. Results from the assay revealed that the inhibitory activity of trypsin-digested rBLG against DPP-IV was similar to that of cBLG. In conclusion, we successfully engineered a strain of NZ9000 that efficiently secretes rBLG, and clearly demonstrated that trypsin-digested rBLG has strong inhibitory activity against DPP-IV enzyme. In future studies, we plan to evaluate the potential of rBLG-secreting NZ9000 as a therapeutic agent for type 2 diabetes.