Abstract: This study aimed to evaluate the inhibitory activity of selenium-containing peptides from selenium-rich black beans against α-amylase and to assess their potential hypoglycemic effects in vitro. Selenium-rich black beans collected from Guangxi, China, were used as raw materials. By combining single-factor optimization, response surface methodology, and peptidomics, four novel peptides were identified and synthesized: Se-MetLP, Se-MetIP, TLTSe-MetLR, and Se-CPSFGQ. Among them, Se-MetIP exhibited the highest inhibitory rate of 70.58%±2.21% at 4 mg/mL, while TLTSe-MetLR showed the lowest half maximal inhibitory concentration (0.47 mg/mL), indicating the most potent effect. To elucidate the mechanism, circular dichroism spectroscopy revealed that peptide binding induced secondary structure remodeling of α-amylase, characterized by increased β-sheet content and reduced β-turns and random coils. Isothermal titration calorimetry and molecular docking confirmed that the binding was exothermic and stabilized mainly by hydrogen bonding and van der Waals forces. Furthermore, functional assays in an insulin-resistant HepG2 cell model demonstrated that Se-MetIP and TLTSe-MetLR significantly enhanced glucose consumption and glycogen synthesis, thereby improving glucose utilization. These findings established a consistent link between peptide-enzyme interactions and cellular metabolic regulation. In conclusion, selenium-containing peptides from black beans not only inhibited α-amylase activity by altering enzyme conformation and stabilizing non-covalent interactions but also promoted glucose metabolism in hepatocytes under insulin resistance. This dual activity highlights their potential as natural hypoglycemic agents and provides a theoretical basis for developing hypoglycemic peptides from black beans.