Nitric oxide (NO) is a potent regulator of both vascular tone and oxygen consumption (Q_O2). Diabetic kidneys have reduced NO availability and increased Q_O2. However, the exact nitric oxide synthase (NOS) isoform regulating hemodynamics, Q_O2 and excretory function in the diabetic kidney remains unclear. We therefore investigated the effects of both specific neuronal (n)NOS inhibition and non-specific NOS inhibition. Renal blood flow (RBF), glomerular filtration rate (GFR), Q_O2, and electrolyte handling were measured in vivo in control and streptozotocin-diabetic rats before and after administration of the specific nNOS inhibitor S- methyl-L-thiocitrulline (1 mg/kg bw/h) or the non- specific NOS inhibitor NG-nitro-L-arginine methylester (10 mg/kg bw/h). Baseline RBF and electrolyte transport efficiency (T_Na/Q_O2) were similar in both groups. Diabetic rats had higher GFR and Q_O2 compared to control. SMTC increased mean arterial pressure (MAP) and decreased RBF in both groups, whereas Q_O2 and T_Na/Q_O2 only decreased in diabetic animals. GFR was unaffected by nNOS inhibition. General NOS inhibition increased MAP and Q_O2 and decreased RBF and GFR in both groups, whereas T_Na/Q_O2 only decreased in the diabetic animals. In conclusion, NO regulates RBF, renal vascular resistance and GFR similarly in both control and diabetic rats. However, RBF is regulated by NO released from nNOS, whereas both NOS blockades affected Q_O2 and T_Na/Q_O2 selectively in the diabetic kidney, indicating a pivotal role for NO in maintaining control of Q_O2. These findings provide mechanistic insights into regulation of the hemodynamics, GFR and Q_O2 in the diabetic kidney, which may contribute to the development of diabetic nephropathy.