Hyperosmotic stress may be initiated during a diverse range pathological circumstances, which in turn results in tissue damage. In this process, the activation of survival signaling, which has the capacity to restore cell homeostasis, determines cell fate. Autophagy is responsible for cell survival and is activated by various pathological stimuli. However, its interplay with hyperosmotic stress and its effect on terminally differentiated cardiac myocytes is unknown. Nuclear factor of activated T-cells 5 (NFAT5), an osmo-sensitive transcription factor, mediates the expression of cell survival associated-genes under hyperosmotic conditions. The present study investigated whether NFAT5 signaling is required in hyperosmotic stress-induced autophagy. It was demonstrated that the presence of a hyperosmotic stress induced an increase in NFAT5 expression, which in turn triggered autophagy through autophagy-related protein 5 (Atg5) activation. By contrast, NFAT5 silencing inhibited DNA damage response 1 protein expression, which then initiated the activation of mammalian target of rapamycin signaling. Therefore, the balance between NFAT5-induced apoptosis and autophagy may serve a critical role in the determination of the fate of cardiomyocytes under hyperosmotic stress. These data suggest that autophagy activation is a beneficial adaptive response to attenuate hyperosmotic stress-induced cell death. Therefore, increasing autophagy through activation of NFAT5 may provide a novel cardioprotective strategy against hyperosmotic stress-induced damage.