We have previously reported that glucosamine protected neonatal rat ventricular myocytes (NRVMs) against ischemia/reperfusion (I/R) injury, and this was associated to an increase in protein O linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus, the initial goal of this study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had similar protective effect as glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5mM) and OGT overexpression increased basal and I/R induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome C. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H₂O₂ and were also associated with an increase in mitochondrial Bcl-2 levels, but had no effect on Bad on Bax levels. Compared to glucosamine and OGT overexpression, NButGT (100 ?M), an inhibitor of O-GlcNAcase, was less protective against I/R and H₂O₂ and did not affect Bcl-2 expression, despite a 5 to 10 fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R induced increase in O-GlcNAc and mitochondrial Bcl-2 and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due in part to enhanced mitochondrial Bcl-2 translocation.