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 Ca imaging and insulin secretion assays. M3G increased intracellular Ca levels in a concentration-dependent manner, specifically targeting β-cells without affecting other pancreatic cell types. It enhanced insulin secretion under both basal (4 mM) and stimulatory (11 mM) glucose conditions while maintaining cell viability at concentrations up to 100 µM. Pharmacological inhibitors revealed that M3G-induced Ca signals resulted from both Ca influx through L-type voltage-dependent calcium channels (L-type VDCCs) and Ca release from the endoplasmic reticulum (ER) via the PLC/IP pathway. Nimodipine, an L-type VDCC blocker, inhibited M3G-induced Ca influx, while U73122 (a PLC inhibitor) and 2-aminoethoxydiphenyl borate (2-APB), an IP receptor blocker, suppressed Ca release from the ER. Additionally, M3G upregulated the expression of key glucose-stimulated insulin secretion (GSIS)-related genes, including Ins1 (insulin), Slc2a2 (GLUT2), and Gck (glucokinase). These findings suggest that M3G stimulates insulin secretion by promoting Ca influx through L-type VDCCs, facilitating Ca 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.