Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca²⁺ signaling, especially Ca²⁺ release from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptor (IP₃R), is considered to be crucial for the regulation of astrocytic functions. Although intraluminal Ca²⁺ dynamics within the ER should be a key determinant of astrocytic Ca²⁺ signaling, technical difficulties have impeded direct analysis. In this study, we developed and used a genetically encoded ER Ca²⁺ indicator, G-CEPIA1er to visualize Ca²⁺ dynamics within the ER in astrocytes in acute brain slices. G-CEPIA1er enabled highly sensitive and selective detection of ER Ca²⁺ release as a decrease in ER Ca²⁺ concentration. This allowed us to reveal a novel mechanism of Ca²⁺ release in astrocytes. Furthermore, we confirmed the crucial role of store-operated Ca²⁺ entry for refilling and maintaining ER Ca²⁺ content after spontaneous and stimulus-induced ER Ca²⁺ release in astrocytes. Collectively, these ER Ca²⁺ visualization studies provide important new insights to Ca²⁺ handling in astrocytes.