Our previous studies demonstrated that caclium-sensing receptor (CaR) stimulation elicited phospholipase C (PLC)-mediated inositol triphosphate (IP(3)) formation, leading to an elevation in [Ca(2+)](i) released from the endo(sarco)plasmic reticulum (ER) to induce ER stress and perturbations of ER function, which cause cardiomyocyte apoptosis during ischemia/reperfusion (I/R). The aim of this study was to determine whether the protection of post-conditioning (PC) from I/R heart injury involved relieving calcium-sensing receptor (CaR)-induced ER stress. Male Wistar rats were subjected to 30 min of ischemia followed by 2 h of reperfusion. The rats were post-conditioned after the 30 min of ischemia by three cycles of 10 s of reperfusion followed by 10 s of ischemia at the onset of reperfusion. Meanwhile, GdCl(3), an activator of CaR, and NPS-2390, a specific inhibitor, were administered. We found that the PC and PC with NPS-2390 groups improved the recovery of cardiac function during reperfusion compared to the IR and PC groups with GdCl(3), respectively. [Ca(2+)](i) and [Ca(2+)](ER) were determined using Fluo-4 AM and Fluo-5N AM, respectively, using laser confocal microscopy. [Ca(2+)](i) was significantly increased, whereas [Ca(2+)](ER) was significantly decreased in the I/R and PC groups with GdCl(3). The rate of apoptotic cells was significantly decreased as shown by TUNEL (Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end labeling) assay in PC and PC with NPS-2390 groups compared to the I/R and PC groups with GdCl(3). In the I/R and PC groups with GdCl(3), the activated fragments of caspase-12, the cleavage products of activating transcription factor 6 (ATF6) and phospho-JNK (c-Jun NH(2)-terminal kinase) were increased compared to the PC and PC with GdCl(3) groups. These results demonstrated that PC could protect the myocardium from I/R injury by inhibiting CaR-induced sarcoplasmic reticulum stress.