Background and aim: As a proinflammatory cytokine, tumor necrosis factor-like weak inducer of apoptosis (TWEAK) participates in the progression of renal fibrosis by binding to its receptor, fibroblast growth factor-inducible 14 (Fn14). However, the effect of Fn14 inhibition on tubular epithelial cell-mediated tubulointerstitial fibrosis remains unclear. This study aimed to elucidate the role of TWEAK/Fn14 interaction in the development of experimental tubulointerstitial fibrosis as well as the protective effect of Fn14 knockdown on proximal tubular epithelial cells. Methods: A murine model of unilateral ureteral obstruction was constructed in both wild-type and Fn14-deficient BALB/c mice, followed by observation of the tubulointerstitial pathologies. Results: Fn14 deficiency ameliorated the pathological changes, including inflammatory cell infiltration and cell proliferation, accompanied by reduced production of profibrotic factors and extracellular matrix deposition. In vitro experiments showed that TWEAK dose-dependently enhanced the expression of collagen I, fibronectin, and α-smooth muscle actin in proximal tubular epithelial cells. Interestingly, TWEAK also upregulated the expression levels of Notch1/Jagged1. Fn14 knockdown and Notch1/Jagged1 inhibition also mitigated the effect of TWEAK on these cells. Conclusions: In conclusion, TWEAK/Fn14 signals contributed to tubulointerstitial fibrosis by acting on proximal tubular epithelial cells. Fn14 inhibition might be a therapeutic strategy for protecting against renal interstitial fibrosis.Keywords: Fibroblast growth factor–inducible 14; Fn14; Proximal tubular epithelial cell; Renal fibrosis; TWEAK; Tumor necrosis factor-like weak inducer of apoptosis; Unilateral ureteral obstruction.