Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis and elevated plasma levels associate with poor cardiovascular and renal outcomes. The dimethylarginine dimethylaminohydrolase enzymes (DDAHs; 1 and 2) metabolise ADMA. A DDAH1 gene variant associates with higher kidney tissue mRNA expression, lower plasma ADMA but counter-intuitively, a steeper rate of eGFR decline. This indicates that renal DDAH1 activity may be deleterious and circulating ADMA does not necessarily reflect the NO-ADMA balance (or severity of disease) within kidney tissue. This study tests the hypothesis that reduced renal DDAH1 activity protects against the progression of kidney function decline, independent of circulating ADMA. Renal DDAH1 expression predominates within the proximal tubule. A novel proximal tubule-specific DDAH1 knock-out (PTD1KO) mouse was developed, which demonstrated tubule-specific dysregulation of ADMA and NO that was not evident systemically. Phenotyping studies in PTD1KO mice did not identify consistent alterations of urinary biochemistry at baseline or after salt loading, however, proteomic analysis revealed significant alterations of urinary peptides at baseline; including down-regulation of uromodulin and collagen. At 12 weeks following folate renal injury, the PTD1KO mouse exhibited less kidney function decline, collagen deposition and pro-fibrotic gene expression (Col12alpha, TGFbeta and ET-1) than controls. Furthermore, ADMA and DDAH1 inhibition reduced tubular sodium and fluid reabsorption in rat microperfusion studies, although studies in PTD1KO mice failed to reproduce this effect. Finally, in vitro studies using a PT cell line and primary PT culture indicated an inhibitory effect of ADMA upon PT cell proliferation. Consistent with recent human genetic studies, these data provide experimental evidence indicating a reduction of renal tubule DDAH1 activity can protect against progressive kidney fibrosis and function decline, independent of plasma ADMA. This work provides novel insights into the role of the NO-ADMA-DDAH axis within the kidney, particularly the tubule.