© 2020 Elsevier Ltd Twenty-two novel dipeptidyl peptidase-IV (DPP-IV) inhibitory peptides (with IC50 values <200 µM) and fifteen novel insulinotropic peptides were identified in a boarfish protein hydrolysate generated at semi-pilot scale using Alcalase 2.4L and Flavourzyme 500L. This was achieved by bioassay-driven semi-preparative reverse phase-high performance liquid chromatography fractionation, liquid chromatography-mass spectrometry and confirmatory studies with synthetic peptides. The most potent DPP-IV inhibitory peptide (IPVDM) had a DPP-IV half maximal inhibitory concentration (IC50) value of 21.72 ± 1.08 µM in a conventional in vitro and 44.26 ± 0.65 µM in an in situ cell-based (Caco-2) DPP-IV inhibition assay. Furthermore, this peptide stimulated potent insulin secretory activity (1.6-fold increase compared to control) from pancreatic BRIN-BD11 cells grown in culture. The tripeptide IPV exhibited potent DPP-IV inhibitory activity (IC50: 5.61 ± 0.20 µM) comparable to that reported for the known DPP-IV inhibitor IPI (IC50: 3.20 µM). Boarfish proteins contain peptide sequences with potential to play a role in glycaemic management in vivo.