Abstract: In order to discover the dipeptidyl peptidase IV (DPP-IV) peptides quickly from Pinctada fucata (P. fucata), in this research, 20 reported DPP-IV inhibitory peptides in the database were used to form a training set and construct pharmacophore models, which were then valuated by test set molecules and fisher random validation. The online website PeptideCutter was employed to perform the virtual hydrolysis with P. fucata meat protein as raw materials. The optimal pharmacophore model (Hypo1) was used to initially screen the 192 low-molecular peptides (the number of amino acids was no more than 5), which were obtained by the virtual hydrolysis, and then the molecular docking was performed for further screening. Furthermore, the potential DPP-IV inhibitory peptides were synthesized using a solid-phase method, and their activities were verified in vitro and the interaction mechanism was analyzed. The results showed that 4 potential DPP-IV inhibitory peptides, namely LPIY, VQDR, PIY, and APSL were screened through pharmacophore combined with molecule docking. Among them, VQDR and PIY did not show inhibitory activity, while LPIY and APSL had strong DPP-IV inhibitory activity, with IC₅₀ values of 521.19 and 258.67 µmol/L respectively. The interaction mechanism indicated both peptides formed multiple hydrogen bonds with the amino acid residues within the active pocket of DPP-IV, and the proline (P) at the second position of the N-terminal constructed Pi-Alkyl interactions with the residues of the active pocket, which promoted the DPP-IV inhibitory activity for LPIY and APSL. The current work would provide a theoretical method for screening DPP-IV inhibitory peptides derived from P. fucata efficiently.