While blockade of the renin angiotensin system (RAS) is beneficial in treating many patients with diabetic nephropathy, some patients show a poor response. We hypothesized that the poor response of RAS blockade is attributed to inability to stimulate endothelial nitric oxide. Recently, we reported that diabetic eNOS knockout (KO) mice develop advanced diabetic nephropathy similar to human disease. Here, we tested the hypothesis that blockade of the RAS would be less beneficial in this model than in diabetic wild-type mice. Both enalapril and telmisartan were less effective at reducing renal injury in diabetic eNOSKO mice compared with diabetic wild-type mice. Blood pressure was only transiently reduced by these treatments in diabetic eNOSKO mice and later returned to levels similar to that of untreated diabetic eNOSKO mice. Serum aldosterone tended to be paradoxically higher with enalapril or telmisartan in diabetic eNOSKO mice, whereas these treatments tended to lower aldosterone in diabetic wild-type mice. The pathogenic role of aldosterone was demonstrated by the evidence that spironolactone significantly reduced blood pressure and prevented renal injury. In addition, a higher dose of enalapril also failed to prevent hypertension and renal injury in diabetic eNOSKO mice. In conclusion, an impaired endothelial NO response could lessen the benefit of RAS inhibition in diabetic renal disease. Aldosterone blockade may provide superior protection in this setting.