Homeostatic calcium ion (Ca(2+)) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca(2+) fluxes. The data showed that endoplasmic reticulum (ER) Ca²⁺ depletion, induced by sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition, resulted to prolonged Ca²⁺ influx and mitochondrial fragmentation within 10 to 30 min. This mitochondrial fragmentation could be prevented in Ca(2+)-free medium or by inhibiting store-operated Ca(2+) entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca(2+) sensors, protected mitochondrial morphology. On the molecular level, ER Ca(2+) depletion, achieved either by removing extracellular Ca(2+) or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca(2+)-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca(2+) fluxes prevent immediate-early cell stress and transcriptional reprogramming.