Macroautophagy is a conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. This study shows that an increase in cytosolic calcium ([Ca²⁺]c) is a potent inducer of macroautophagy. Various calcium mobilizing agents, such as vitamin D3 compounds, ionomycin, ATP, and thapsigargin, inhibit the mammalian target of rapamycin (mTOR), a negative regulator of autophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β) and AMP-activated protein kinase (AMPK), and inhibited by ectopic Bcl-2 located in the endoplasmic reticulum (ER), where it lowers [Ca²⁺]ER and attenuates agonist-induced Ca²⁺ fluxes. Thus, an increase in [Ca²⁺]c serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2. The study also shows that Ca²⁺ mobilizing agents induce autophagy via a signaling pathway involving CaMKK-β, AMPK, and mTOR, and that ER-localized Bcl-2 effectively inhibits this pathway. The findings suggest that Ca²⁺ plays a key role in autophagy signaling and that ER-localized Bcl-2 can inhibit autophagy by modulating Ca²⁺ homeostasis.Macroautophagy is a conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. This study shows that an increase in cytosolic calcium ([Ca²⁺]c) is a potent inducer of macroautophagy. Various calcium mobilizing agents, such as vitamin D3 compounds, ionomycin, ATP, and thapsigargin, inhibit the mammalian target of rapamycin (mTOR), a negative regulator of autophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca²⁺/calmodulin-dependent kinase kinase-β (CaMKK-β) and AMP-activated protein kinase (AMPK), and inhibited by ectopic Bcl-2 located in the endoplasmic reticulum (ER), where it lowers [Ca²⁺]ER and attenuates agonist-induced Ca²⁺ fluxes. Thus, an increase in [Ca²⁺]c serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2. The study also shows that Ca²⁺ mobilizing agents induce autophagy via a signaling pathway involving CaMKK-β, AMPK, and mTOR, and that ER-localized Bcl-2 effectively inhibits this pathway. The findings suggest that Ca²⁺ plays a key role in autophagy signaling and that ER-localized Bcl-2 can inhibit autophagy by modulating Ca²⁺ homeostasis.