Increases in the cytosolic Ca²⁺ concentration ([Ca²⁺]c) in pancreatic β-cells mediate glucose-stimulated insulin secretion. It has been suggested that increases in the mitochondrial Ca²⁺ concentration ([Ca²⁺]m) are also involved in insulin secretion promoting mitochondrial ATP production. However, [Ca²⁺]m dynamics during glucose stimulation require further clarification. Using mitochondrial Ca²⁺ indicator CEPIA2mt, we here analyzed glucose-stimulated [Ca²⁺]m dynamics in a pancreatic β-cell line MIN6. During glucose stimulation, [Ca²⁺]c showed oscillatory changes with intervals of 2–3 min. [Ca²⁺]m, on the other hand, showed very subtle and unexpected changes: it decreased with an increase in [Ca²⁺]c and increased with a decrease in [Ca²⁺]c. However, upon shRNA-mediated knockdown of MICU1, a gatekeeper protein of mitochondrial calcium uniporter (MCU), [Ca²⁺]m increased in phase with [Ca²⁺]c oscillations having much greater amplitudes than those in control cells. Despite the remarkable increase in [Ca²⁺]m dynamics, glucose-stimulated [Ca²⁺]c dynamics remained almost the same. These results indicate that [Ca²⁺]m is tightly regulated by MICU1 during glucose stimulation, and that increases in [Ca²⁺]m above the baseline level seem not to be necessary for the generation of glucose-stimulated [Ca²⁺]c oscillations.