Background: Dysregulation of lipid metabolism has been implicated to be important in the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, the role of 12-hydroxyeicosatetraenoic acid (12S-HETE), a derivative of the arachidonic acid, is not well defined in the pathogenesis of IPF. Methods: The level of 12S-HETE was measured in human plasma (IPF = 76, control = 40) via liquid chromatography-mass spectrometry (LC-MS/MS). Western blot was performed to evaluate the protein expression of collagen type I (collagen I), alpha-smooth muscle actin (α-SMA) and arachidonate 12-lipoxygenase (ALOX12), lipoxygenase- type enzyme that produces 12S-HETE, in human lung tissues and 12S-HETE-treated human lung fibroblast (MRC-5) cells. The function of ALOX12 was confirmed by ALOX12 inhibitor (ML355) and siRNA in MRC-5 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the mRNA expression of epithelial-mesenchymal transition (EMT) markers such as CDH1, E-cadherin coding gene, SNAIL, SLUG and VIM, vimentin coding gene, in 12S-HETE-treated human lung bronchial alveolar epithelial (BEAS-2B) cells. Results: The level of 12S-HETE was increased in plasma of IPF patients compared with controls. The protein levels of collagen I, α-SMA, and ALOX12 were increased in human lung tissues of IPF patients compared with controls. The level of ALOX12 was increased in TGF-β1-treated MRC-5 cells. Moreover, 12S-HETE increased the protein expression of collagen I and α-SMA in MRC- 5 cells, and ML355 and ALOX12 siRNA decreased the protein levels of collagen I, and α-SMA in MRC-5 cells. 12S-HETE increased the mRNA expression levels of SNAIL, SLUG and VIM but, decreased the that of E-cadherin in BEAS-2B cells. Conclusions: Our findings suggest that 12S-HETE may have pro-fibrotic effects by activation of fibroblasts and be implicated as a potential therapeutic target in IPF.