The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca(2+) homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca(2+) homeostasis, and the ER Ca(2+) store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca(2+) leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca(2+) depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca(2+) leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca(2+) permeability and subsequent ER stress can lead to development of a novel therapeutic approach.