Overactivity of renal sympathetic nerves and nitric oxide (NO) deficiency occur in hypertensive chronic kidney disease (CKD). In sheep with hypertensive CKD and NO deficiency, renal denervation (RDN) reduces blood pressure and improves kidney function (glomerular filtration rate). We hypothesized that this improvement in glomerular filtration rate after RDN is associated with increased NO bioavailability. In this study, glomerular filtration rate response to systemic inhibition of NOS (NO synthase) was examined in healthy and CKD sheep at 2 and 30 months after a sham (intact nerves) or RDN procedure. Basal urinary total nitrate (nitrate+nitrite) excretion was examined at 2 and 30 months, and kidney protein expression of endothelial and neuronal NOS was assessed at 30 months. Urinary nitrate+nitrite in CKD-RDN and healthy sheep was ≈50% to 70% greater than in CKD-intact. During NOS inhibition, the fall in glomerular filtration rate in CKD-RDN sheep was ≈20% greater than in CKD-intact. These effects in CKD-RDN sheep were similar to those in healthy sheep. Endothelial NOS protein expression was lower in CKD-intact sheep compared with healthy sheep and compared with CKD-RDN. In summary, RDN normalizes NO bioavailability and restores contribution of NO to renal hemodynamics in CKD. These changes may promote improvements in kidney function and sustained blood pressure lowering after RDN in hypertensive CKD.