The modification of proteins with O-linked N-acetylglucosamine (O-GlcNAc) is increasingly recognized as an important posttranslational modification that modulates cellular function. Recent studies suggested that augmentation of O-GlcNAc levels increase cell survival following stress. Salidroside, one of the active components of Rhodiola rosea, shows potent anti-hypoxia property. In the present study, we reported the cardioprotection of salidroside from ischemia and reperfusion. Cardiomyocytes were exposed to 4 h of ischemia and 16 h of reperfusion, and then cell viability, apoptosis, glucose uptake, ATP levels and cytosolic Ca2+ concentration were determined, and O-GlcNAc levels were assessed by Western blotting. Salidroside (80 uM) was added 24 h before ischemia/reperfusion was induced. Treatment with salidroside markedly improved cell viability from 64.7 ± 4.5% to 85.8 ± 3.1%, decreased lactate dehydrogenase (LDH) release from 38.5 ± 2.1% to 21.2 ± 1.7%, reduced cell apoptosis from 27.2 ± 3.2% to 12.2 ± 1.9%, significantly improved cardiomyocytes glucose uptake by 1.7-fold and increased O-GlcNAc levels by 1.6-fold, as well as reducing cytosolic Ca2+ concentration compared to untreated cells following ischemia/reperfusion. Furthermore, the improved cell survival and the increase in O-GlcNAc with salidroside were attenuated by alloxan, an inhibitor of O-GlcNAc transferase. These results suggested that salidroside significantly enhances glucose uptake and increases protein O-GlcNAc levels and this is associated with decreased cardiomyocytes injury following ischemia/reperfusion.