Abstract: Objective: The purpose was to optimize the enzymatic hydrolysis process of Pinctada martensii flesh by in vitro dipeptidyl peptidase (DPP-IV) inhibition rate and glucose consumption of human liver cancer cell (HepG-2) insulin resistance model, and to analyze the hypoglycemic peptides. Methods: The glucose consumption of human liver cancer cell (HepG-2) insulin resistance model and the in vitro dipeptidyl peptidase (DPP-IV) inhibition rate were evaluated as indicators for comparison. The enzymatic hydrolysis effects of acid protease, alkaline protease, neutral protease, compound protease, trypsin, pepsin, and papain on Pinctada martensii flesh were compared and screened. The effect of enzymolysis temperature, material-liquid ratio, enzyme dosage, and enzymolysis time on the hypoglycemic activity of enzymatic hydrolysate were analyzed by single factor experiments and orthogonal test. The best enzymatic hydrolysis process was determined. Nano-LC Q Exactive Orbitrap Mass Spectrometry (Nano LC-MS/MS) was used to analyze the composition of peptides in enzymatic hydrolysis. Results: The product glucose consumption and DPP-IV inhibition rate of acid protease hydrolysis were better than other proteases. The optimal process conditions for the preparation of the Pinctada martensii flesh were: 45 ℃, 3 h, material-liquid ratio 1:3.5 (w:w), and 1000 U/g protamex, under these conditions, the glucose consumption was 37.53% and the DPP-IV inhibition rate was 74.21%. The molecular weight (M_W) of 93.88% of peptides from Pinctada martensii flesh under the best technology was below 2000 Da. 22.63% of the peptides were hydrophobic peptides, and 74.92% of the peptides contained at least one hydrophobic amino acid at the N-terminus. Conclusion: The Pinctada martensii flesh hypoglycemic peptides prepared by the enzymatic method could inhibit the activity of DPP-IV and reduced insulin resistance to play a hypoglycemic effect, which had a certain guiding significance for the development of functional foods.