BACKGROUND: Renal fibrosis is a key pathological process in chronic kidney disease (CKD), characterized by excessive extracellular matrix (ECM) deposition and epithelial-mesenchymal transition (EMT). Current treatment strategies have limited efficacy, necessitating the exploration of novel therapeutic agents. Eicosapentaenoic acid (EPA), a bioactive marine-derived omega-3 polyunsaturated fatty acid, has shown promise in modulating fibrosis-related signaling pathways. OBJECTIVES: This study investigated the potential of EPA in mitigating renal fibrosis through the regulation of the transforming growth factor-β1 (TGF-β1)/Smad3/ILK pathway and its effects on ECM remodeling and EMT suppression in human kidney epithelial cells. MATERIALS AND METHODS: Human Kidney-2 (HK-2) cells were subjected to albumin-induced EMT and treated with EPA, either alone or in combination with the β-catenin inhibitor LF3. The expression levels of key EMT markers (E-cadherin, N-cadherin, vimentin), ECM regulators (MMPs and TIMPs), and fibrosis-related signaling molecules (TGF-β1, Smad3, ILK) were assessed using immunofluorescence, ELISA, RT-qPCR, and Western blot analysis. RESULTS: EPA treatment significantly inhibited EMT by downregulating α-SMA, N-cadherin, vimentin, and active β-catenin while restoring E-cadherin expression (p < 0.05). ECM remodeling was evident through increased MMP-1, MMP-3, and MMP-9 expression and decreased TIMP-1 and TIMP-2 levels. Furthermore, EPA reduced TGF-β1, ILK, and phosphorylated Smad3 protein levels, an effect enhanced by LF3 co-treatment. CONCLUSION: EPA shows preliminary potential as an antifibrotic agent in vitro by targeting the TGF-β1/Smad3/ILK pathway to regulate ECM remodeling and EMT suppression in renal fibrosis. This study provides insights into the EPA's application in medical biotechnology, particularly for CKD management.