Oxygen and nitrogen derived free radicals and oxidant plays important role in the pathogenesis of diabetic endothelial dysfunction. Present study was aimed to analyze the role of superoxide (O2.-) on endothelial impairment and its correlation with constitutive nitrous oxide synthetase isoenzymes in the penis of the long-term diabetic rat. Wistar albino rats (Rattus norvegicus) were randomly divided into two groups i.e. control (received 0.1M of citrate buffer) and diabetes (received single dose of Streptozotocin at 60 mg/kg in 0.01M citrate buffer through i.p.). At the end of 120th day, animals were sacrificed by overdose of anesthesia (thiopentone sodium 40mg/kg b.wt.) and immediately penile tissue were dissected collected and used for the various analyses like protein and mRNA expression of iNOS, nNOS, eNOS and MnSOD. Small pieces of tissues were used for histological and immunohistological analyses (iNOS, nNOS, eNOS and MnSOD). In-situ detection O2.- using dihydroethedine was performed on fresh tissue sections. Results showed significant increase in the production of superoxide and significant decrease in the levels of NOS isoensymes (iNOS – p< 0.001, eNOS – p< 0.001 and nNOS – p< 0.001) and Mn SOD (p< 0.001) in the penis of the diabetic animal. Levels of mRNA expression of NOS isoenzymes (iNOS – p< 0.001, eNOS – p< 0.01 and nNOS – p< 0.001) and MnSOD (p< 0.001) were significantly deceased in diabetic rat penis. Histological observations showed increase in endothelial thickening of the diabetic rat penis. These observations indicates that the elevated levels of O2.- mediates endothelial dysfunction and this increased levels of O2.- might be due to the impaired mitochondrial antioxidant defense system or/and alterations in the electron transport system. This might down-regulate the mRNA expression of NOS isoenzymes or up-regulate the degradation of these enzymes. Thus understanding the mitochondrial pathophysiological source of O2.- and mRNA expression of these enzymes under hyperglycemic condition would give more insight into ensuing erectile dysfunction.