Metabolic syndrome is a significant risk factor for the development of hepatocellular carcinoma (HCC). 25-HC (25-hydroxycholesterol) synthesized from cholesterol plays an important role in lipid metabolism. However, the functions and mechanism of 25-HC in HCC remain largely unknown. In this study, we demonstrated that 25-HC promoted HCC cells migration and intrahepatic and extrahepatic metastasis while did not affect the cells proliferation and apoptosis. Mechanistically, the promotive effect of 25-HC was through up-regulation of Toll-like receptor 4 (TLR4) dependent fatty acid binding protein 4 (FABP4). Inhibition of FABP4 hindered 25-HC-induced cells migration and metastasis. Moreover, up-regulation of FABP4 was observed in HCC tissues from database analysis. In summary, our study reveals the effects and mechanism of 25-HC/TLR4/FABP4 axis in promoting HCC metastasis, which provides novel avenue for therapeutic intervention against HCC progression.