Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca²⁺]_c) is a potent inducer of macroautophagy. Various Ca²⁺ mobilizing agents (vitamin D₃ compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, 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 and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca²⁺]_ER and attenuates agonist-induced Ca²⁺ fluxes. Thus, an increase in the [Ca²⁺]_c serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.