AMP-activated protein kinase (AMPK) has emerged as a pertinent stress-activated protein kinase shown to have substantial cardioprotective capabilities against myocardial ischemia/reperfusion injury. When activated during ischemia, AMPK produces effects that have been demonstrated to be beneficial to the myocardium by means of increasing GLUT4 translocation and glucose uptake, activating eNOS, decreasing apoptosis, and increasing autophagic flux. However, the molecular basis behind the regulation of AMPK activity in the ischemic and reperfused heart remains incompletely understood. Recent evidence implicates the role of Sestrin2 in the AMPK signaling pathway and it is hypothesized that Sestrin2 plays an influential role during myocardial ischemia in order to promote AMPK activation. Sestrin2 protein was found to be expressed in adult cardiomyocytes and accumulated in the heart during ischemic conditions. Sesn2 KO mice were used to determine the importance of Sestrin2 during I/R injury. Sesn2 KO cardiac phenotype analysis indicated no significant difference in left ventricular function as measured by a pressure-volume loop catheter. When wild type (WT) and Sesn2 KO mice were subjected to in vivo I/R, myocardial infarct size was significantly greater in Sesn2 KO compared to WT hearts. Similarly, Langendorff perfused hearts indicated exacerbated post-ischemic contractile function in Sesn2 KO hearts when compared to WT. Ischemic AMPK activation was found to be impaired in the Sesn2 KO hearts. Immunoprecipitation of Sestrin2 demonstrated an association with AMPK. Moreover, LKB1, a major AMPK upstream kinase was associated with the Sestrin2-AMPK complex in a time-dependent manner during ischemia while this interaction was nearly abolished in Sesn2 KO hearts. Thus, Sestrin2 plays an important role in cardioprotection against I/R injury by acting as an LKB1-AMPK scaffold to initiate AMPK activation during ischemic insults.