Chronic kidney disease (CKD) is a worldwide public health problem which is caused by repeated injuries to the glomerulus or renal tubules. Renal fibrosis commonly accompanies CKD and it is histologically characterized by excessive deposition of extracellular matrix proteins, such as fibronectin and collagen I, in interstitial areas. Indirect in vivo experimental data suggest that renal asymmetric dimethylarginine (ADMA) exerts anti-fibrotic activity in CKD. In the current study, we aimed to demonstrate that renal ADMA has a direct effect on fibrosis in vivo. Normal saline (NS), ADMA, non-sense control (NC) siRNA, Ddah1 siRNA or Ddah2 siRNA were administered into the kidney through the left ureter in a mouse model of unilateral ureteral obstruction (UUO). UUO kidneys were harvested at day 1 or day 7. Western blotting was performed to assess the expression of ADMA, DDAH1, DDAH2 and expression of fibrotic markers such as fibronectin, collagen I, aSMA, pSmad3, and CTGF. Masson’s trichrome staining was used to further evaluate renal fibrosis. We observed that intrarenal administration of ADMA increased the renal accumulation of ADMA and attenuated renal fibrosis at day 1 and day 7. Knockdown of Ddah1 or Ddah2 increased the amount of ADMA in UUO kidneys and inhibited the expression of fibrotic proteins at day 1 and day 7, which was further confirmed by Masson staining. Thus, our in vivo data suggest that renal ADMA exerts direct anti-fibrotic effects in a mouse model of UUO.This article is protected by copyright. All rights reserved.