Chronic kidney disease (CKD) often progresses to renal fibrosis, which is characterized by excessive extracellular matrix deposition and is also linked to ferroptosis. The present study investigated how TGF-β1 induces ferroptosis and thereby contributes to renal tubular epithelial cell fibrosis. Bioinformatics was employed to identify the differentially expressed genes relevant to renal fibrosis. An in vitro TGF-β1-induced fibrosis model of HK-2 cells was established, and the cell shape index was calculated. Fer-1, NAC, and PD98059 were utilized for targeted intervention, and their mechanisms were verified by transducing cells with WISP1-targeting shRNA lentivirus. Cell morphology was examined under a microscope, and cells were collected to determine the levels of ferroptosis-related factors (Fe2+, MDA, GSH, and LPO). Western blotting was performed to measure the levels of ERK1/2, WISP1, and ferroptosis indicators (GPX4 and hyperoxidized PRDX4). Flow cytometry was performed to determine the ROS levels and the rate of cell ferroptosis. TGF-β1 induced the transformation of HK-2 cells into fibroblast-like cells, leading to increased ROS levels, activation of the ERK1/2-WISP1 signaling pathway, and upregulation of ferroptosis and fibrosis-related factors. However, these effects could be effectively inhibited through pretreatment with Fer-1, NAC, and PD98059 individually, which further validated the involvement of the ERK1/2-WISP1 signaling pathway. In addition, WISP1 knockdown suppressed the cell transformation into fibroblast-like cells as well as the ferroptosis process, thereby reducing the expression levels of ferroptosis and fibrosis-related factors. The present study substantiated the process through which TGF-β1 elicits the production of ROS and triggers ferroptosis via the ERK1/2-WISP1 signaling pathway to facilitate the development of renal tubular epithelial cell fibrosis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10616-025-00719-5.