Pressure overload associated with hypertension is an important pathological factor leading to heart remodeling and ultimately heart failure partially due to cardiomyocyte apoptosis. Here we show that endogenous NO signaling plays a critical role in mechanical stretch-induced cardiomyocyte apoptosis. Mechanical stretch induced elevated expression of both eNOS and inducible NO synthase (iNOS) and increased synthesis of NO. A sustained increase in iNOS expression was also found in hearts of hypertensive rats in vivo. Blockade of NO signaling by inhibitors of NOS (L-NAME and AMT) or downstream guanylyl cyclase (ODQ) strongly inhibited stretch-induced apoptosis, mitochondria depolarization, and cytochrome c release, suggesting that NO is required in stretch-induced cardiomyocyte apoptosis. The expression of iNOS, but not eNOS, was blocked by L-NAME and ODQ, indicating that the iNOS induction is NO dependent. The initial elevation of NO is likely due to Ca(2+)-dependent activation of eNOS because elimination of intracellular calcium by EGTA-AM inhibited both iNOS induction and NO elevation. Other calcium signaling inhibitors (nifedipine, ryanodine, thapsigargin, and ionic gadolinium) also attenuated the initial NO elevation. These data indicate that mechanical signals initiate Ca(2+)-dependent NO synthesis, which is further amplified by activation of NO-induced iNOS expression, to regulate cardiomyocyte apoptosis.