Diabetes is associated with increased production of reactive oxygen species leading to decreased nitric oxide (NO) bioavailability and intrarenal hypoxia. Plasma L-arginine concentration, the substrate for NO synthesis, is reduced in diabetes and we therefore hypothesis that L-arginine or L-citrulline supplementation improve NO production and thereby protects kidney function in diabetes. Glomerular filtration rate (GFR), total renal blood flow (RBF), in vivo oxygen consumption (QO2), cortical oxygen tension (PO2) and urinary protein excretion were evaluated in control and streptozotocin-diabetic male Sprague-Dawley rats with and without L-arginine or L-citrulline supplementation for the 21-day duration of diabetes. GFR was increased in untreated (2.6±0.2 ml/min/kidney) and L-arginine treated diabetics (2.3±0.2 ml/min/kidney) compared to controls (1.2 ml/min/kidney). However, L-citrulline administration to diabetics prevented the glomerular hyperfiltration (1.4±0.1 ml/min/kidney). RBF was not affected by diabetes or any of the treatments, resulting in increased calculated filtration fraction (FF) in untreated diabetics compared to controls (0.39±0.04 vs. 0.27±0.02), which was prevented by L-citrulline treatment to diabetics (0.25±0.04). The increased urinary protein excretion in untreated diabetics (142±25 vs. 75±7 mg/min/kidney) was also prevented by L-citrulline treatment (67±7 mg/min/kidney). The diabetes-induced intrarenal tissue hypoxia, resulting from increased QO2, was unaffected by any of the treatments. In conclusion, L-citrulline, but not L-arginine supplementation prevents diabetes-induced glomerular hyperfiltration by reducing the FF, which also prevents the increased urinary protein excretion commonly associated with the progression of diabetic nephropathy.