The calcium-sensing receptor (CaR) is a G protein-coupled receptor. The CaR stimulation elicits phospholipaseC-mediated inositol triphosphate formation, leading to an elevation in the level of intracellular calcium released from endoplasmicreticulum (ER). Depletion of ER Ca2+ leads to ER stress, which is thought to induce apoptosis. Intracellular calciumoverload-induced apoptosis in cardiac myocytes during hypoxia–reoxygenation (H ?Re) has been demonstrated. However, thelinks between CaR, ER stress and apoptosis during H?Re are unclear. This study hypothesized that the CaR could induceapoptosis in neonatal rat cardiomyocytes during H?Re via the ER stress pathway. Neonatal rat cardiomyocytes were subjectedto 3 hr of hypoxia, followed by 6 hr of reoxygenation. CaR expression was elevated and the number of apoptotic cells wassignificantly increased, as shown by transferase-mediated dUTP nick end-labelling, with exposure to CaCl2, a CaR activator,during H?Re. The intracellular calcium concentration was significantly elevated and the Ca2+ concentration in the ER wasdramatically decreased during H?Re with CaCl2; both intracellular and ER calcium concentrations were detected by laserconfocal microscopy. Expression of GRP78 (glucose-regulated protein 78), the cleavage products of ATF6 (activating transcriptionfactor 6), phospho-PERK [pancreatic ER kinase (PKR)-like ER kinase], the activated fragments of caspase-12, andphospho-JNK (c-Jun NH2-terminal kinase) were increased following exposure to CaCl2 during H?Re. Our results confirmedthat the activated CaR can induce cardiomyocyte apoptosis via ER stress-associated apoptotic pathways during H? Re.