Ca2+ may trigger programmed cell death (apoptosis) and regulate death-specific enzymes. Therefore, the development of strategies to control Ca2+ homeostasis may represent a potential approach to prevent or enhance cell apoptosis. To test this hypothesis, the plasma membrane Na/Ca exchanger (NCX1.7 isoform) was stably overexpressed in insulin-secreting tumoral cells. NCX1.7 overexpression increased apoptosis induced by endoplasmic reticulum (ER) Ca2+-ATPase inhibitors, but not by agents increasing intracellular calcium concentration ([Ca2+]i), through the opening of plasma membrane Ca2+-channels. NCX1.7 overexpression reduced the rise in [Ca2+]i induced by all agents, depleted ER Ca2+ stores, sensitized the cells to Ca2+-independent proapoptotic signaling pathways, and reduced cell proliferation by ∼40%. ER Ca2+ stores depletion was accompanied by the activation of the ER-specific caspase (caspase-12), and the activation was enhanced by ER Ca2+-ATPase inhibitors. Hence, Na/Ca exchanger overexpression, by depleting ER Ca2+ stores, triggers the activation of caspase-12 and increases apoptotic cell death. By increasing apoptosis and decreasing cell proliferation, overexpression of Na/Ca exchanger may represent a new potential approach in cancer gene therapy. On the other hand, our results open the way to the development of new strategies to control cellular Ca2+ homeostasis that could, on the contrary, prevent the process of apoptosis that mediates, in part, β-cell autoimmune destruction in type 1 diabetes.