It is well accepted that chronic hyperglycemia is an important risk factor for myocardial infarction and importantly, both acute and chronic hyperglycemia trigger several biochemical and electrophysiological changes resulting an impaired cardiac contractile function. Hypeglycemia first initiates repeated acute changes in cellular metabolism, and then followed by cumulative long-term changes in macromolecules. The long-term changes include mainly a big amount of increases in the production of reactive oxygen species, ROS, which then induce a diabetic tissue/cell damage in several target organs including heart. Marked changes in cytosolic free Zn 2+ occurs during contractile activity and Zn 2+ movements are controlled by changes in cytosolic free Ca 2+ , being highly sensitive to cellular oxidative-state in cardiomyocytes. In here, we examined whether cellular antioxidant-defence enhancement preserves cardiac dyfunction via regulation of both diastolic free Zn 2+ and Ca 2+ . The N -acetyl cysteine (NAC)-treatment of diabetic rats led to a balanced oxidant/antioxidant level in both heart and circulation and prevented the altered cellular redox-state. It also prevented diabetes-induced tissue damage and markedly increased diastolic function with marked normalizations in the resting levels of both diastolic free Zn 2+ and Ca 2+ . Both in vivo and in vitro NAC-treatment of diabetic samples prevented the altered kinetic parameters of transient changes in both free Zn 2+ and Ca 2+ under electrical stimulation, as well as spatio-temporal properties of both local changes of free Zn 2+ and Ca 2+ (sparks) in resting cells. Biochemical analysis demonstrated that NAC-treatment of diabetic rats also antagonized hyperphosphorylation of cardiac ryanodine receptors (RyR2) and significantly restored depleted protein levels of both RyR2 and calstabin2. Incubation of cardiomyocytes with 10-µM ZnCl 2 exerted hyperphosphorylation in RyR2 as well as higher phosphorphorylations in both PKA and CaMKII in a concentration-dependent manner, similar to hyperglycemia. Our present data also showed that a subcellular oxidative stress marker, NF-κB can be activated if the cells are exposed directly to Zn 2+ . We thus for the first time report that an enhancement of antioxidant-defence in diabetics via directly targeting heart, seems to prevent diastolic dysfunction, associated with normalization of RyR2 macromolecular-complex, and thereby prevention of both Zn 2+ and Ca 2+ leaks via leading to normalization of both diastolic free Zn 2+ and Ca 2+ in cardiomyocytes (Supported with Tubitak SBAG-214S254.