Perturbance of endoplasmic reticulum (ER) function, either by the mutant proteins not folding correctly, or by an excessive accumulation of proteins in the organelle, will lead to the unfolded protein response (UPR) or ER overload response (EOR). The signal-transducing pathways for UPR have been identified, whereas the pathway for EOR remains to be elucidated. Our previous study demonstrated that the overexpression of reticulon 3 (RTN3, also named HAP, homologue of ASY protein) caused apoptosis with the depletion of ER Ca2+ stores. In present research, we characterized RTN3 as a novel EOR-induced protein, triggering the apoptotic signals through the release of ER Ca2+ and the elevation of cytosolic Ca2+. Our studies showed that overexpressed RTN3 induced EOR, eliciting ER-specific apoptosis with activation of caspase-12 and mitochondrial dysfunction through ER Ca2+ depletion and the sustained elevation of cytosolic Ca2+. Furthermore, we demonstrated that overexpressed RTN3 and stimuli that activate both EOR and UPR, not UPR only, were able to induce up-regulation of inducible nitric oxide synthase (iNOS) in HeLa cells through ER Ca2+ release and reactive oxygen intermediates (ROIs), resulting in endogenous calcium-dependent nitric oxide protecting cells against ER specific apoptosis, which suggested that the nitric oxide and iNOS represented a likely protective response to EOR, not the UPR. These results supported that the release of ER Ca2+ stores triggered the initial signal-transducing pathways for EOR induced by overexpressed RTN3. © 2005 Wiley-Liss, Inc.