Aims: Increased O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins has been implicated in the adverse effects of diabetes on the heart, although this has typically been based on models of severe hyperglycemia. Diabetes has also been associated with dysregulation of autophagy, a critical cell survival process; however, little is known regarding autophagy in the diabetic heart or whether this is influenced by O-GlcNAcylation or hemodynamic stress. Main methods: Young male rats were assigned to control (12% kcal fat/19% protein/69% carbohydrate), high fat diet (60/19/21%) and type 2 diabetic (high fat diet + low dose streptozotocin) groups for 8 weeks, followed by sham or pressure overload surgeries; animals were sacrificed 8 weeks after surgery. Key findings: A modest increase in arterial pressure resulted in no significant effects on cardiac function in control or high fat groups, while diabetic hearts exhibited contractile dysfunction and increased apoptosis and scar formation. Immunoprecipitation studies revealed, for the first time, that Beclin-1, which plays a critical early role in autophagy, and the anti-apoptotic Bcl-2, are targets for O-GlcNAcylation. Interestingly, we also found that cardiomyocytes isolated from type 2 diabetic db/db mice exhibited a blunted autophagic response and this was at least partially reversed by inhibiting glucose entry into the hexosamine biosynthesis pathway, which regulates O-GlcNAc synthesis. We also found that acutely augmenting O-GlcNAc levels in non-diabetic cardiomyocytes mimicked the effects of diabetes by blunting autophagic signaling. Significance: These data suggest that O-GlcNAc-mediated inhibition of autophagy may contribute to the abnormal response of diabetic hearts to hemodynamic stress. © 2012 Elsevier Inc.