Glucose-induced stimulation of β-cells results in oscillatory changes in cytosolic Ca²⁺ concentration, which lead to insulin secretion and may also cause flux of Ca²⁺ into the mitochondrial matrix. Although recent reports suggest that Ca²⁺ uptake by mitochondria via mitochondrial Ca²⁺ uniporter (MCU) facilitates insulin secretion, mitochondrial Ca²⁺ dynamics in β-cells require further clarification. Using the recently developed CEPIAmt Ca²⁺ indicators, we analyzed mitochondrial Ca²⁺ dynamics in high glucose-stimulated β-cells. Contrary to our expectation, oscillatory increases in cytosolic Ca²⁺ concentrations in response to high glucose stimulation induced minimal changes in mitochondrial Ca²⁺ concentration. Upon shRNA-mediated knockdown of MICU1, one of the essential regulators of MCU, we observed markedly increased oscillatory changes in the mitochondrial Ca²⁺ concentration. In addition, MICU1-depleted β-cells showed a significant decrease in glucose-induced insulin secretion. These results indicate that MICU1 limits mitochondrial Ca²⁺ uptake in β-cells, and such tight regulation of mitochondrial Ca²⁺ signals is required to maintain insulin secretion. To further understand the physiological and pathophysiological significance of the MICU1-dependent mitochondrial Ca²⁺ regulations in insulin secretion, we plan to use β-cell-specific MICU1 knockout genome-edited mice.