O-linked β-N-acetylglucosamine (O-GlcNAc) is an inducible, dynamically cycling and reversible post-translational modification of Ser/Thr residues of nucleocytoplasmic and mitochondrial proteins. We recently discovered that O-GlcNAcylation confers cytoprotection in the heart via attenuating the formation of mitochondrial permeability transition pore (mPTP) and the subsequent loss of mitochondrial membrane potential. Because Ca(2+) overload and reactive oxygen species (ROS) generation are prominent features of post-ischemic injury and favor mPTP formation, we ascertained whether O-GlcNAcylation mitigates mPTP formation via its effects on Ca(2+) overload and ROS generation. Subjecting neonatal rat cardiac myocytes (NRCMs, n ≥ 6 per group) to hypoxia, or mice (n ≥ 4 per group) to myocardial ischemia reduced O-GlcNAcylation, which later increased during reoxygenation/reperfusion. NRCMs (n ≥ 4 per group) infected with an adenovirus carrying nothing (control), adenoviral O-GlcNAc transferase (adds O-GlcNAc to proteins, AdOGT), adenoviral O-GlcNAcase (removes O-GlcNAc to proteins, AdOGA), vehicle or PUGNAc (blocks OGA; increases O-GlcNAc levels) were subjected to hypoxia-reoxygenation or H(2)O(2), and changes in Ca(2+) levels (via Fluo-4AM and Rhod-2AM), ROS (via DCF) and mPTP formation (via calcein-MitoTracker Red colocalization) were assessed using time-lapse fluorescence microscopy. Both OGT and OGA overexpression did not significantly (P > 0.05) alter baseline Ca(2+) or ROS levels. However, AdOGT significantly (P < 0.05) attenuated both hypoxia and oxidative stress-induced Ca(2+) overload and ROS generation. Additionally, OGA inhibition mitigated both H(2)O(2)-induced Ca(2+) overload and ROS generation. Although AdOGA exacerbated both hypoxia and H(2)O(2)-induced ROS generation, it had no effect on H(2)O(2)-induced Ca(2+) overload. We conclude that inhibition of Ca(2+) overload and ROS generation (inducers of mPTP) might be one mechanism through which O-GlcNAcylation reduces ischemia/hypoxia-mediated mPTP formation.