Resveratrol is a natural phytophenol that exhibits cardioprotective effects. This study was designed to elucidate the mechanisms by which resveratrol protects against diabetes-induced cardiac dysfunction. Normal control (m-Lepr(db)) mice and type 2 diabetic (Lepr(db)) mice were treated with resveratrol orally for 4 wk. In vivo MRI showed that resveratrol improved cardiac function by increasing the left ventricular diastolic peak filling rate in Lepr(db) mice. This protective role is partially explained by resveratrol's effects in improving nitric oxide (NO) production and inhibiting oxidative/nitrative stress in cardiac tissue. Resveratrol increased NO production by enhancing endothelial NO synthase (eNOS) expression and reduced O(2)(·-) production by inhibiting NAD(P)H oxidase activity and gp91(phox) mRNA and protein expression. The increased nitrotyrosine (N-Tyr) protein expression in Lepr(db) mice was prevented by the inducible NO synthase (iNOS) inhibitor 1400W. Resveratrol reduced both N-Tyr and iNOS expression in Lepr(db) mice. Furthermore, TNF-α mRNA and protein expression, as well as NF-κB activation, were reduced in resveratrol-treated Lepr(db) mice. Both Lepr(db) mice null for TNF-α (db(TNF-)/db(TNF-) mice) and Lepr(db) mice treated with the NF-κB inhibitor MG-132 showed decreased NAD(P)H oxidase activity and iNOS expression as well as elevated eNOS expression, whereas m-Lepr(db) mice treated with TNF-α showed the opposite effects. Thus, resveratrol protects against cardiac dysfunction by inhibiting oxidative/nitrative stress and improving NO availability. This improvement is due to the role of resveratrol in inhibiting TNF-α-induced NF-κB activation, therefore subsequently inhibiting the expression and activation of NAD(P)H oxidase and iNOS as well as increasing eNOS expression in type 2 diabetes.