Kidney fibrosis is the hallmark of chronic kidney disease (CKD) progression, whereas no effective anti-fibrotic therapies exist. Recent evidence has shown that tubular ferroptosis contributes to the pathogenesis of CKD with persistent proinflammatory and profibrotic responses. We previously reported that natural flavonol fisetin alleviated septic acute kidney injury and protected against hyperuricemic nephropathy in mice. In this study, we investigated the therapeutic effects of fisetin against fibrotic kidney disease and the underlying mechanisms. We established adenine diet-induced and unilateral ureteral obstruction (UUO)-induced CKD models in adult male mice. The two types of mice were administered fisetin (50 or 100 mg·kg·d, i.g.) for 3 weeks or 7 days, respectively. At the end of the experiments, the mice were euthanized, and blood and kidneys were gathered for analyzes. We showed that fisetin administration significantly ameliorated tubular injury, inflammation, and tubulointerstitial fibrosis in the two types of CKD mice. In mouse renal tubular epithelial (TCMK-1) cells, treatment with fisetin (20 μM) significantly suppressed adenine- or TGF-β1-induced inflammatory responses and fibrogenesis, and improved cell viability. By quantitative real-time PCR analysis of ferroptosis-related genes, we demonstrated that fisetin treatment inhibited ferroptosis in the kidneys of CKD mice as well as in injured TCMK-1 cells, as evidenced by decreased ACSL4, COX2, and HMGB1, and increased GPX4. Fisetin treatment effectively restored ultrastructural abnormalities of mitochondrial morphology and restored the elevated iron, the reduced GSH and GSH/GSSG as well as the increased lipid peroxide MDA in the kidneys of CKD mice. Notably, abnormally high expression of the ferroptosis key marker ACSL4 was verified in the renal tubules of CKD patients (IgAN, MN, FSGS, LN, and DN) as well as adenine- or UUO-induced CKD mice, and in injured TCMK-1 cells. In adenine- and TGF-β1-treated TCMK-1 cells, ACSL4 knockdown inhibited tubular ferroptosis, while ACSL4 overexpression blocked the anti-ferroptotic effect of fisetin and reversed the cytoprotective, anti-inflammatory, and anti-fibrotic effects of fisetin. In summary, we reveal a novel aspect of the nephroprotective effect of fisetin, i.e. inhibiting ACSL4-mediated tubular ferroptosis against fibrotic kidney diseases.