Objective: This study aims to investigate the effect of metformin and p38-MAPK inhibitor (SB203580) combination on myocardial ischemia/reperfusion injury in hyperglycemic condition and type 2 diabetic rat Methods: Rat cardiac myoblast (H9c2) cells were induced hyperglycemic condition by using 33 mM of D-Glucose solution in the presence and absence of metformin, or SB203580, or the combination between metformin and SB203580. Cells were subjected to simulated ischemia/reperfusion (sI/R) injury. Cell viability was determined by MTT cell survival assay. In addition Wistar rat and Goto-Kakizaki (GK) rat were treated with metformin, SB203580, the combination between metformin and SB203580 for 4 weeks. The fasting blood glucose level and Hemoglobin A1c (HbA1c) level were assessed. An ex vivo experiments, the hearts were performed by Langendorff perfusion system at 30 min stabilization, 30 min ischemia, and 90 min reperfusion. The infarct size was determined by TTC staining. Results: An in vitro experiments, incubation of 33 mM D-glucose solution in H9c2 cell enhanced the sensitivity of I/R injury in H9c2 cell (p < 0.05). Treatment with drugs in hyperglycemic subjected to sI/R condition were significantly increased cell viability. The results in animal study showed that GK rats were significantly increased in fasting blood glucose level and HbA1c level and both parameters were significantly decreased by drugs treatment (p < 0.05). An ex vivo experiment, the stimulation of I/R injury in GK rat hearts were significantly increased infarct size (p < 0.05). Treatment with metformin, SB203580, the combination between 2 drugs were significantly decreased infarct size (p < 0.05). Conclusion: Treatment with the combination between metformin and SB203580 showed the cardio-protective effects on hyperglycemia subjected to I/R injury by reducing cell death (in vitro) and infarct size (ex vivo).