The endoplasmic reticulum (ER) plays indispensable roles as a source and sink of Ca2+ in both neuronal and glial cells in the central nervous system (CNS). Although intraluminal Ca2+ dynamics within the ER is crucial for neuronal/glial physiology and pathophysiology, 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 neurons and astrocytes. We found ER Ca2+ replenishment by intraluminal Ca2+ diffusion throughout the ER of dendrites and spines, indicating that the ER functions as a pipeline to redistribute stored Ca2+ in neurons. Furthermore, 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 from the ER in astrocytes. Collectively, these ER Ca2+ visualization studies provide important new insights to Ca2+ handling in neurons and astrocytes, which should be the basis of CNS pharmacology in regard to Ca2+ signaling.