Abstract: Mulberry leaves were a rich source of high-quality protein and possess the potential to yield bioactive peptides. This study identified novel α-glucosidase inhibitory peptides from mulberry leaf proteolytic products based on peptidomics identification and the structure-activity relationships of active peptides. Through molecular docking and network pharmacology predictions, the anti-diabetic mechanisms of these novel α-glucosidase inhibitory peptides were elucidated. The results indicated that two novel α-glucosidase inhibitory peptides were identified: ANSTGSRL (IC₅₀=0.314 mg/mL) and LAKLLHSK (IC₅₀=0.183 mg/mL). LAKLLHSK exhibited a higher content of β-sheet structure and a lower content of α-helix and random coil structures compared to ANSTGSRL. The gastrointestinal digestion process enhanced the release of α-glucosidase inhibitory activity for both peptides, achieving over 85% enzyme inhibition. Molecular docking analysis revealed that the two peptides tightly bound to the active residue sites of α-glucosidase (Val335, Val334, Pro230 and Gly228), primarily through multiple hydrogen bonds and hydrophobic interactions, thereby exerting their α-glucosidase inhibitory activity. Network pharmacology predictions indicate that two peptides, ANSTGSRL and LAKLLHSK, can interact with over ten key targets, including MAPK1, CASP3, STAT1, ERBB2 and AKT3. KEGG pathway enrichment analysis revealed that the anti-diabetic pathway associated with these peptides involves multiple complex signaling pathways, including the TNF, cAMP, PI3K-Akt, and MAPK pathways, as well as insulin resistance. The novel mulberry leaf peptides ANSTGSRL and LAKLLHSK exhibit strong α-glucosidase inhibitory activity and potential glucose regulation effects. This study could provide a theoretical reference for the high-value utilization of mulberry leaf protein resources.