Renal interstitial fibrosis (RIF) is a main pathological process in chronic kidney disease (CKD). Demethylzeylasteral (DML), a major component of Tripterygium wilfordii Hook. f., has anti-renal fibrosis effects. However, its mechanism of action remains incompletely understood.The present study was designed to comprehensively examine the effects of DML on RIF and the underlying mechanisms.Pathological experiments were performed to determine the therapeutic effect of DML on a mouse model of UUO-induced RIF. To determine the novel mechanisms underlying the therapeutic effects of DML against RIF, a comprehensive transcriptomics analysis was performed on renal tissues, which was further verified by a series of experiments.Pathological and immunohistochemical staining showed that DML inhibited UUO-induced renal damage and reduced the expression of fibrosis-related proteins in mice. Transcriptomic analysis revealed that the partial subunits of mitochondrial complex (MC) I and II may be targets by which DML protects against RIF. Furthermore, DML treatment reduced mitochondrial reactive oxygen species (ROS) levels, consequently promoting ATP production and mitigating oxidative stress-induced injury in mice and cells. Notably, this protective effect was attributed to the inhibition of MC I activity, suggesting a crucial role for this specific complex in mediating the therapeutic effects of DML against RIF.This study provides compelling evidence that DML may be used to treat RIF by effectively suppressing mitochondrial oxidative stress injury mediated by MC I. These findings offer valuable insights into the pharmacological mechanisms of DML and its potential clinical application for patients with CKD.Copyright © 2024. Published by Elsevier B.V.