Sestrin2 (Sesn2) is a stress-inducible protein that plays a critical role in the response to ischemic stress. We recently recognized that Sesn2 may protect the heart against ischemic insults by reducing the generation of reactive oxygen species (ROS). After 45 min of ischemia followed by 24 h of reperfusion, myocardial infarcts were significantly larger in Sesn2 KO hearts than in wild-type hearts. Isolated cardiomyocytes from wild-type hearts treated with hypoxia and reoxygenation (H/R) stress showed significantly greater Sesn2 levels, compared with normoxic hearts (p < 0.05). Intriguingly, the administration of adeno-associated virus 9-Sesn2 into Sesn2 knockout (KO) hearts rescued Sesn2 protein levels and significantly improved the cardiac function of Sesn2 KO mice exposed to ischemia and reperfusion. The rescued levels of Sesn2 in Sesn2 KO hearts significantly ameliorated ROS generation and the activation of ROS-related stress signaling pathways during ischemia and reperfusion. Moreover, the rescued Sesn2 levels in Sesn2 KO cardiomyocytes improved the maximal velocity of cardiomyocyte shortening by H/R stress. Rescued Sesn2 levels also improved peak height, peak shortening amplitude, and maximal velocity of the re-lengthening of Sesn2 KO cardiomyocytes subjected to H/R. Finally, the rescued Sesn2 levels significantly augmented intracellular calcium levels and reduced the mean time constant of transient calcium decay in Sesn2 KO cardiomyocytes exposed to H/R. Overall, these findings indicated that Sesn2 can act as an endogenous antioxidant to maintain intracellular redox homeostasis under ischemic stress conditions.