Overexpression of the inducible isoform of the enzyme nitric oxide synthase (iNOS) has been associated to pathological processes in the kidney. Ethanol consumption induces the renal expression of iNOS; however, the contribution of this enzyme to the deleterious effects of ethanol in the kidney remains elusive. We examined whether iNOS plays a role in the renal dysfunction and oxidative stress induced by ethanol consumption. With this purpose, male C57BL/6 wild-type (WT) or iNOS-deficient (iNOS–/–) mice were treated with ethanol (20% v/v) for 10 weeks. Treatment with ethanol increased the expression of Nox4 as well as the concentration of thiobarbituric acid reactive substances and the levels of tumor necrosis factor α in the renal cortex of WT but not iNOS–/– mice. Augmented serum levels of creatinine and increased systolic blood pressure were found in WT and iNOS–/– mice treated with ethanol. WT mice treated with ethanol showed increased production of reactive oxygen species and myeloperoxidase activity, but these responses were attenuated in iNOS–/– mice. We concluded that iNOS played a role in ethanol-induced oxidative stress and pro-inflammatory cytokine production in the kidney. These are mechanisms that may contribute to the renal toxicity induced by ethanol.