Rationale: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of L-arginine (L-arg), the substrate for endothelial nitric oxide synthase (eNOS), failed to improve vascular function. L-Citrulline (L-cit) supplementation not only increases L-arg synthesis, but also inhibits cytosolic arginase I, a competitor of eNOS for the use of L-arg, in the vasculature. Aims: To investigate whether L-cit treatment reduces DN in streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice and rats and to study its effects on arginase II (ArgII) function, the main renal isoform. Methods: STZ-C57BL6 mice received L-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and L-cit-treated STZ-rats were evaluated. Results: L-Citrulline exerted protective effects in kidneys of STZ-rats, and markedly reduced urinary albumin excretion, tubulo-interstitial fibrosis, and kidney hypertrophy, observed in untreated diabetic mice. Intriguingly, L-cit treatment was accompanied by a sustained elevation of tubular ArgII at 16 weeks and significantly enhanced plasma levels of the anti-inflammatory cytokine IL-10. Diabetic ArgII knock out mice showed greater blood urea nitrogen levels, hypertrophy, and dilated tubules than diabetic wild type (WT) mice. Despite a marked reduction in collagen deposition in ArgII knock out mice, their albuminuria was not significantly different from diabetic WT animals. L-Cit also restored nitric oxide/reactive oxygen species balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, L-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1β and IL-12(p70) generation in the human proximal tubular cells. Conclusion: L-Citrulline supplementation established an anti-inflammatory profile and significantly preserved the nephron function during T1D. © 2013 Romero, Yao, Sridhar, Bhatta, Dou, Ramesh, Brands, Pollock, Caldwell, Cederbaum, Head, Bagi, Lucas and Caldwell.