NADPH oxidase Nox4-derived reactive oxygen species (ROS) play important roles in renal fibrosis. Our previous study demonstrated that intermedin (IMD) alleviated unilateral ureteral obstruction (UUO)-induced renal fibrosis by inhibition of ROS. However, the precise mechanisms remain unclear. Herein, we investigated the effect of IMD on Nox4 expression and NADPH oxidase activity in rat UUO model, and explored if these effect were achieved through cAMP-PKA pathway, the important post-receptor signal transduction pathway of IMD, in TGF-β1-stimulated rat proximal tubular cell (NRK-52E). Renal fibrosis was induced by UUO. NRK-52E was exposed to rhTGF-β1 to establish an in vitro model of fibrosis. IMD was overexpressed in the kidney and in NRK-52E by IMD gene transfer. We studied UUO-induced ROS by measuring dihydroethidium levels and lipid peroxidation end-product 4-hydroxynonenal expression. Nox4 expression in the obstructed kidney of UUO rat or in TGF-β1-stimulated NRK-52E was measured by quantitative RT-PCR and Western blotting. We analyzed NADPH oxidase activity using a lucigenin-enhanced chemiluminescence system. We showed that UUO-stimulated ROS production was remarkably attenuated by IMD gene transfer. IMD overexpression inhibited UUO-induced up-regulation of Nox4 and activation of NADPH oxidase. Consistent with in vivo results, TGF-β1-stimulated increase in Nox4 expression and NADPH oxidase activity was blocked by IMD. In NRK-52E, these beneficial effects of IMD were abolished by pretreatment with N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide hydrochloride (H-89), a PKA inhibitor, and mimicked by a cell-permeable cAMP analog dibutyl-cAMP. Our results indicate that IMD exerts anti-oxidant effects by inhibition of Nox4, and the effect can be mediated by cAMP-PKA pathway.