•Protaetia brevitarsis larva protein was used for enzymatically hydrolysis.•Four peptides with Angiotensin-I-converting enzyme (ACE) inhibiting activity were identified.•In silico molecular docking analysis reveal that the peptides were hydrogen bonded with the ACE active site.•The four peptides promoted NO production in HUVECs. Many angiotensin-I-converting enzyme (ACE) inhibitory peptides are used to prevent and manage hypertension. In this study, ACE inhibitory peptides were isolated from an insect protein that is attracting attention for it potential antihypertensive activity. Protaetia brevitarsis larva protein was enzymatically hydrolyzed by Flavourzyme®, and the hydrolysate was shown to inhibit ACE. Subsequent fractionation, using ultrafiltration and gel permeation chromatography followed by liquid chromatography-tandem mass spectrometry analysis, identified four previously unknown peptides with significant ACE inhibition characteristics (Ser-Tyr, Pro-Phe, Tyr-Pro-Tyr, and Trp-Ile). The highest inhibition activity observed for Trp-Ile. These peptides stimulated production of NO in human umbilical vein endothelial cells and, based on molecular docking analysis, exerted their inhibitory effects via hydrogen bonding with the ACE receptor active site. Thus, the identified peptides can be considered as promising candidates for ACE inhibition and have potential to be used as functional food ingredients.