Amygdalus communis L. is rich in high-quality protein and its natural peptides have potential in controlling and preventing diseases. The objective of this study was to evaluate the hypoglycemic activity of bioactive peptides obtained from Amygdalus communis L. protein by synergistic hydrolysis of trypsin and protamex. After ultrafiltration and purification, active peptide B4 with small molecular weight in four hypoglycemic active peptides showed the best inhibitory activities on α-amylase and α-glucosidase. Moreover, insulin resistance model was established with HepG2 cells to study the hypoglycemic mechanism of active peptide B4. The experimental results confirmed that the active peptide B4 was able to up-regulate GYS1 gene mRNA expression and down-regulate PEPCK, G6Pase and GP gene mRNA expression. Meanwhile, active peptide B4 significantly increased the glucose consumption and glycogen synthesis in IR-HepG2. This research enhances the additional value of nut proteins and contributes to the development of potential functional products to expand their application in the food field.