<p>Malvidin-3-glucoside (M3G), an anthocyanin found in blueberries and grapes, shows promise as a natural anti-diabetic agent. However, its effect on insulin secretion and its underlying mechanisms remains unclear. This study investigated the impact of M3G on β-cells (INS-1) through real-time Ca²⁺ imaging and insulin secretion assays. M3G increased intracellular Ca²⁺ levels in a concentration-dependent manner, specifically targeting β-cells without affecting other pancreatic cell types. It enhanced insulin secretion under both basal (4&#xa0;mM) and stimulatory (11&#xa0;mM) glucose conditions while maintaining cell viability at concentrations up to 100&#xa0;µM. Pharmacological inhibitors revealed that M3G-induced Ca²⁺ signals resulted from both Ca influx through L-type voltage-dependent calcium channels (L-type VDCCs) and Ca²⁺ release from the endoplasmic reticulum (ER) via the PLC/IP₃ pathway. Nimodipine, an L-type VDCC blocker, inhibited M3G-induced Ca²⁺ influx, while U73122 (a PLC inhibitor) and 2-aminoethoxydiphenyl borate (2-APB), an IP₃ receptor blocker, suppressed Ca²⁺ release from the ER. Additionally, M3G upregulated the expression of key glucose-stimulated insulin secretion (GSIS)-related genes, including <i>Ins1</i> (insulin), <i>Slc2a2</i> (GLUT2), and <i>Gck</i> (glucokinase). These findings suggest that M3G stimulates insulin secretion by promoting Ca²⁺ influx through L-type VDCCs, facilitating Ca²⁺ release from the ER, and upregulating GSIS-related genes. M3G holds promise as a natural anti-diabetic agent by enhancing insulin secretion and supporting β-cell function.</p>