Insulin secretion from pancreatic β-cells, which is regulated by intracellular Ca²⁺ signals, plays a crucial role in the control of blood glucose levels. Although Ca²⁺ signals in β-cells have been analyzed using in vitro/ex vivo preparations including cell lines, isolated β-cells and isolated islets, in vivo Ca²⁺ imaging has proved to be challenging. Thus, while activities of β-cells in living animals are under the influence of autonomic nervous system, hormones and other substances, Ca²⁺ signals of β-cells under physiological conditions have remained elusive. We here report an in vivo β-cells Ca²⁺ imaging method using a transgenic mouse line expressing a genetically encoded Ca²⁺ indicator YC-Nano50. Using the method, we succeeded in analyzing Ca²⁺ signals in β-cells in laparotomized mice under anesthesia, and observed synchronized Ca²⁺ oscillations in β-cells within individual islets. Furthermore, simultaneous Ca²⁺ imaging in multiple islets revealed synchronized Ca²⁺ oscillations among islets, which is the basis for pulsate insulin secretion that underlies the oscillation of blood insulin level. The present method is expected to help us gain deeper insight into the regulation of insulin secretion and diabetes.