Abnormal mechanical load, as seen in hypertension, is found to induce heart cell apoptosis, yet the signaling link between cell stretch and apoptotic pathways is not known. Using an in vitro stretch model mimicking diastolic pressure stress, here we show that Ca2+. signaling participates essentially in the early stage of stretch-induced apoptosis. In neonatal rat cardiomyocytes, the moderate 20% stretch resulted in tonic elevation of intracellular free Ca2+ ([Ca2+](i)). Buffering [Ca2+](i) by EGTA-AM, suppressing ryanodine-sensitive Ca2+ release. and blocking L-type Ca2+ channels all prevented the stretch-induced apoptosis as assessed by phospbatidylserine exposure and nuclear fragmentation. Notably, Ca2+ suppression also prevented known stretch-activated apoptotic events, including caspase-3/-9 activation, mitochondrial membrane potential corruption, and reactive oxygen species production, suggesting that Ca2+ signaling is the upstream of these events. Since [Ca2+](i) did not change without activating mechanosensitive Ca2+ entry, we conclude that stretch-induced Ca2+ entry, via the Ca2+-induced Ca2+ release mechanism, plays an important role in initiating apoptotic signaling during mechanical stress. Published by Elsevier Inc.