An increase in oxidative stress is suggested to be the main cause in Doxorubicin (Dox) -induced cardiotoxicity. However, there is now evidence that activation of inducible nitric oxide synthase (iNOS) and nitrosative stress are also involved. The role of Vitamin C (Vit C) in the regulation of nitric oxide synthase (NOS) and reduction of nitrosative stress in Dox-induced cardiotoxicity is unknown. The present study investigated the effects of Vit C in the mitigation of Dox-induced changes in the levels of nitric oxide (NO), NOS activity, protein expression of NOS isoforms and nitrosative stress as well as cytokines TNFα and IL-10 in isolated cardiomyocytes. Cardiomyocytes isolated from adult Sprague Dawley rats were segregated into four groups: i) control; ii) Vit C (25 µM); iii) Dox (10 µM); and iv) Vit C + Dox. Dox caused significant increase in the generation of superoxide radical (O₂^-.), peroxynitrite and NO and these effects of Dox were blunted by Vit C. Dox increased the expression of iNOS and altered protein expression as well as activation of endothelial NOS (eNOS). These changes were prevented by Vit C. Dox-induced increase in the ratio of monomeric/dimeric eNOS, promoting the production of O₂^-., which was prevented by Vit C by increasing the stability of dimeric form of eNOS. Vit C protected against Dox-induced increase in TNFα as well as a reduction in IL-10. These results suggest that Vit C provides cardioprotection by reducing oxidative/nitrosative stress and inflammation via a modulation of Dox-induced increase in the NO levels and NOS activity.