Objective Tetrahydrobiopterin (BH4) is a multifunctional co-factor having potential to regulation mitochondria function including biogenesis and oxidative phosphorylation. Diabetic cardiomyopathy is the major cause of mortality and morbidity in diabetes mellitus patients. Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. The aim of this study is to test the mitochondria mediated therapeutic potential of BH4 in the treatment of diabetic cardiomyopathy Methods Fifty weeks aged LETO and OLETF rats were used as control and type 2 diabetes animal models respectively. Onset of diabetes was confirmed by intravenous glucose tolerance test (IGTT). Randomly selected OLETFs were administrated BH4 20mg/kg/day bolus i.p. during 2 weeks (OLETF/BH4). Results Administration of BH4 did not altered IGTT, body weight or blood component of OLETF. Echocardiography revealed dilated dysfunction in OLETF and OLETF/BH4 model compared to LETO. BH4 treatment significantly increased left ventricular contractility in OLETF resulting in enhanced ejection fraction and fractional shortening. Mitochondrial membrane potential, electron transport chain complex activity and ATP concentration were decreased in OLETF model and BH4 treatment successfully restored those. Interestingly, increased oxidative stress in OLETF heart tissues were significantly attenuated by BH4 treatment. RT-PCR and western blot analysis showed that BH4 treatment restored several mitochondrial protein alterations in type 2 diabetic heart. Conclusion These results suggest that BH4 has therapeutic potential which corrected mitochondrial dysfunction resulting enhancement of LV contractility in diabetic cardiomyopathy.