Black　shark　skin　was　enzymatically　hydrolyzed　by　protease　to　produce　antioxidant　peptides.　The　mechanism　of　action　of　the　peptides　was　explored.　The　hydrolysis　process　was　optimized　based　on　1,1-diphenyl-2-picrylhydrazyl　(DPPH)radical　scavenging　capacity　and　also　by　taking　into　account　degree　of　hydrolysis.　The　antioxidant　peptides　were　separated　and　purified　by　reversed-phase　high　performance　liquid　chromatography　(RP-HPLC).　The　amino　acid　sequences　were　identified　by　electrospray　ionization　quadrupole-time　of　fl　ight　mass　(ESI　Q-TOF　MS),　and　the　in　vitro　antioxidant　activity　of　the　purified　peptides　was　analyzed.　The　optimal　hydrolysis　parameters　were　determined　as　follows:　use　of　alkaline　protease　as　the　best　enzyme,　enzyme　dosage　311　U/mL,　substrate　concentration　3%,　temperature　45,　pH　8.0　and　time　4.9　h.　The　hydrolysate　obtained　with　a　degree　of　hydrolysis　of　14.21%　under　the　optimized　conditions　scavenged　77.61%　of　DPPH　radical.　The　hydrolysate　was　separated　into　26　fractions.　F19　was　found　to　possess　the　strongest　DPPH　radical　scavenging　activity.　The　total　ion　current　peak　with　higher　antioxidant　activity　(at　10.02　min　retention　time)　was　selected　for　mass　spectrometric　(MS)　and　tandem　mass　spectrometric　(MS/MS)　analysis.　The　amino　acid　sequence　of　F19　was　determined　as　Pro-Gly-Gly-Thr-Met.　F19　at　1.0　mg/mL　scavenged　75.01%　of　DPPH　radical,　and　its　oxygen　radical　absorption　capacity(ORAC,　calculated　as　Trolox　equivalent)　was　2　490.01　μmol/g.　Pro　and　Met　residues　played　a　leading　role　in　the　antioxidant　activity　of　F19.　This　study　provides　a　theoretical　basis　for　understanding　the　antioxidant　mechanism　and　the　structureactivity　relationship　of　the　pentapeptide　derived　from　black　shark　skin.　©　2019,　China　Food　Publishing　Company.　All　right　reserved.