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.