Intracellular Ca²⁺ signal plays an essential role in insulin secretion from pancreatic β-cells. Recent reports suggest that Ca²⁺ release from the endoplasmic reticulum (ER) through cholinergic receptor stimulation mediated by parasympathetic nerves contributes to Ca²⁺ signals in β-cells. However, how the Ca²⁺ release from the ER shapes the intracellular Ca²⁺ signal remains elusive due to limitations in the methods for direct visualization analysis. We recently developed transgenic mouse lines expressing a genetically encoded cytosolic Ca²⁺ indicator, YC-Nano50, or an ER Ca²⁺ indicator, CEPIA specifically in β-cells. We successfully observed periodic oscillations of both cytosolic and ER Ca²⁺ signals evoked by high glucose in isolated pancreatic islets. We also confirmed a cholinergic agonist-induced decrease in ER Ca²⁺, i.e. Ca²⁺ release from the ER. Surprisingly, during a high glucose condition, short-term cholinergic agonist application induced a transient suppression of cytosolic Ca²⁺ level to the extent comparable with the resting level, despite the release of Ca²⁺ from the ER. Our results suggest that parasympathetic nerves mediate suppressive regulation of Ca²⁺ signaling. Further analysis is required to reveal the physiological roles and underlying mechanisms of this unexpected Ca²⁺ suppression.