In　this　work,　four　novel　angiotensin　I-converting　enzyme　inhibitory　(ACEI)　peptides　were　isolated　and　identified　from　peony　(Paeonia　suffruticosa　Andr.)　seed　hydrolysate　(PSH)　by　consecutive　chromatography　and　UPLC-ESI-QTOF-MS/MS.　The　IC50　values　of　HWS　(0.640　±0.016　mg/mL)　and　VLSGF　(0.328　±　0.040　mg/mL)　were　significantly　(p　<　0.05)　lower　than　those　of　the　other　two　peptides.　The　Lineweaver-Burk　plots　showed　that　VLSGF　is　competitive　inhibition　against　ACE　and　HWS　acted　as　a　mixed-type　inhibitor.　Meanwhile,　the　results　of　molecular　docking　showed　hydrogen　interactions　between　VLSGF/HWS　and　ACE.　In　human　endothelial　cells,　the　pretreatments　of　VLSGF　and　HWS　could　increase　the　production　of　nitric　oxide　(NO)　and　the　expression　of　endothelial　nitric　oxide　synthase　(eNOS).　In　addition,　they　also　decreased　the　reactive　oxygen　species　(ROS)　production.　These　results　indicated　that　it　may　be　possible　to　use　peony　seed　protein　hydrolysate　to　create　ingredients　for　functional　foods.　©　2022　The　Authors