Astrocytes regulate various brain functions, for which Ca2+ release from the endoplasmic reticulum (ER) often play crucial roles. Because astrocytic ER Ca2+ release is robust and frequent, the ER Ca2+ refilling mechanism should be critical for ongoing Ca2+ signaling in astrocytes. In this study, we focused on the putative functional significance of store-operated Ca2+ entry (SOCE) in ER Ca2+ refilling. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in astrocytes in acute cortical slices to directly monitor the decrease and recovery of ER Ca2+ concentration upon spontaneous or norepinephrine-induced Ca2+ release. Inhibition of SOCE significantly slowed the recovery of ER Ca2+ concentration after Ca2+ release in astrocytes. This delayed recovery resulted in a prolonged decrease in the ER Ca2+ content in astrocytes with periodic spontaneous Ca2+ release, followed by the attenuation of cytosolic Ca2+ responses upon Ca2+ release. Therefore, our results provide direct evidence for the physiological significance of SOCE in ER Ca2+ refilling after ER Ca2+ release. (C) 2019 Elsevier Inc. All rights reserved.