Renal interstitial fibrosis (RIF) is a common pathway for progression of chronic kidney disease (CKD) to end-stage renal disease. In our previous study, it has been provided that renalase can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction (UUO) rats. The other mechanism of renalase to alleviate renal fibrosis should be explored. In this study, we evaluated the role of oxidative stress in UUO rats and epithelial-mesenchymal transition (EMT) in human proximal renal tubular epithelial (HK-2) cells and examined the association of renalase. Oxidative stress could induce EMT and fibrosis in HK-2 cells. In the UUO model, oxidative stress occurred and maintained at a high level leading to RIF. Administration of renalase by adenovirus significantly attenuated oxidative stress and further in vitro study showed that renalase can exert anti-fibrosis by inhibiting oxidative stress. Our results prompt another mechanism of anti-fibrotic renal protection of renalase, which may be partly associated with inhibiting oxidative stress. These data provided another theoretical basis that supplementation with exogenous renalase may be a promising strategy for slowing or halting the progression of CKD.