Clinical trials demonstrate the regenerative potential of cardiac stem cell (CSC) therapy in the postinfarcted heart. Despite these encouraging preliminary clinical findings, the basic biology of these cells remains largely unexplored. The principal requirement for cell transplantation is to effectively prime them for survival within the unfavorable environment of the infarcted myocardium. In the adult mammalian heart, the β-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a unique post-translational modification that confers cardioprotection from various otherwise lethal stressors. It is not known whether this signaling system exists in CSCs. In this study, we demonstrate that protein O-GlcNAcylation is an inducible stress response in adult murine Sca-1+/lin- CSCs and exerts an essential prosurvival role. Posthypoxic CSCs responded by time-dependently increasing protein O-GlcNAcylation upon reoxygenation. We used pharmacological interventions for loss- and gain-of-function, that is, enzymatic inhibition of O-GlcNAc transferase (OGT) (adds the O-GlcNAc modification to proteins) by TT04, or inhibition of OGA (removes O-GlcNAc) by thiamet-G (ThG). Reduction in the O-GlcNAc signal (via TT04, or OGT gene deletion using Cre-mediated recombination) significantly sensitized CSCs to posthypoxic injury, whereas augmenting O-GlcNAc levels (via ThG) enhanced cell survival. Diminished O-GlcNAc levels render CSCs more susceptible to the onset of posthypoxic apoptotic processes via elevated poly(ADP-ribose) polymerase cleavage due to enhanced caspase-3/7 activation, whereas promoting OGlcNAcylation can serve as a pre-emptive antiapoptotic signal regulating the survival of CSCs. Thus, we report the primary demonstration of protein O-GlcNAcylation as an important prosurvival signal in CSCs, which could enhance CSC survival prior to in vivo autologous transfer. © AlphaMed Press.