Intracellular Ca²⁺ signals regulate insulin secretion from pancreatic β-cells. Although recent reports suggest that mitochondria influence insulin secretion through their Ca²⁺ uptake activity, mitochondrial Ca²⁺ dynamics in β-cells have remained elusive due to limitations in the method for mitochondrial Ca²⁺ visualization. Using recently developed high-performance intraorganellar Ca²⁺ indicators, CEPIA, we here analyzed high glucose-induced mitochondrial Ca²⁺ dynamics in an insulinoma cell line MIN6. Unexpectedly, high glucose-stimulation evoked only limited mitochondrial Ca²⁺ signals. Moreover, shRNA-mediated knockdown of mitochondrial calcium uptake 1 (MICU1), which is one of the essential regulators of mitochondrial Ca²⁺ uptake, significantly enhanced high glucose-induced mitochondrial Ca²⁺ signals. Furthermore, MICU1 knockdown caused a decrease in high glucose-induced insulin secretion. These results suggest that MICU1 negatively regulates mitochondrial Ca²⁺ uptake to maintain insulin secretion. Further analysis is required to clarify the relationship between mitochondrial Ca²⁺ uptake and insulin secretion.