Intracellular Ca²⁺ signaling in pancreatic β-cells and liver hepatocytes contributes to the homeostatic regulation of living organisms by triggering the secretion of glycemia-controlling hormones and metabolic processes. Ca²⁺ signals in these cells have been studied in imaging analysis using ex vivo preparations. Unlike the case of ex vivo analysis, β-cells and hepatocytes in vivo are under the influence of the autonomic nervous system, hormones, nutrients, and other bioactive substances. Therefore, Ca²⁺ activities in β-cells and hepatocytes under physiological conditions remain elusive. We here report in vivo Ca²⁺ activities of β-cells and hepatocytes using transgenic mouse lines expressing a ratiometric Ca²⁺ indicator protein in β-cell- or hepatocyte-specific manner. In vivo imaging analysis of these mice enabled us to visualize spatiotemporal patterns of Ca²⁺ signals that were not predicted by ex vivo Ca²⁺ imaging analysis. These results and our future study are expected to clarify the detailed mechanisms for homeostatic regulation by β-cells and hepatocytes.