In response to injury, cells and tissues remodel their cellular environment to repair damaged structures and if necessary to initiate apoptosis. This process, known as the cellular stress response, includes robust and dynamic changes in the modification of intracellular proteins by monosaccharides of O-linked b-N-acetylglucosamine (O-GlcNAc). Acute enhancment of O-GlcNAcylation reduces apoptosis and necrosis in both in vivo and in vitro models of injury that include cardiac ischemia reperfusion injury (I/R injury). Although enhanced O-GlcNAcylation suppresses the hallmarks of I/R injury, the molecular mechanism(s) by which O-GlcNAc mediates cardioprotection are largely uncharacterized. The present study sought to determine whether a causal relationship exists between injury-induced O-GlcNAcylation and autophagy, as both cellular processes are induced by ischemic preconditioning and promote cardioprotection. We engineered forms of the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that catalyze the addition and removal of O-GlcNAc, that enable the regulation of protein O-GlcNAcylation with biologically inert small molecules. We demonstrate that enhancing wildtype OGT expression is associated with inceased autophagic flux and reduces cell death of H9C2 cells challenged with oxidative stress. Consistent with these data, pharmocological augmentation of O-GlcNAcylation raises autophagosome levels in murine hearts and H9C2 cells. Autophagosome accumulation corresponds to increased proautophagic signaling as indicated by AMP-activated protein kinase (AMPK) and ULK1 activation. The increase in autophagy associated with elevated O-GlcNAcylation was curtailed by AMPK inhibition or deletion, indicating that O-GlcNAc signaling regulates autophagy in part through AMPK. As such, we assessed the O-GlcNAcylation state of these autophagy regulators. AMPKa and ULK1 are O-GlcNAc-modified or associate with O-GlcNAc-modified proteins in a stress-dependent manner. Together, our data suggests that O-GlcNAc can positively regulate autophagy at multiple points along the pathway.