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The intracellular fatty acid binding proteins (FABPs) are a well-conserved family which functions as lipid chaperones and also crucial mediators of cellular signaling.However, the biologic functions and mechanism of FABPs remain largely unknown.Here we report that FABP5 acts as a key regulator on milk fat synthesis in bovine mammary epithelial cells (BMECs).We showed that overexpression of FABP5 in BMECs enhanced the gene expression of SREBP-lc to promote milk fat synthesis whereas knock down of FABP5 had the opposed effects.FABP5 existed in the cytoplasm, and estrogen (E) and methionine (Met) upregulated the expression of FABP5.Furthermore, we confirmed that FABP5 mediates the upregulation of milk fat synthesis induced by Met and E, through stimulation of the gene expression of SREBP-1 c but not PPARβ/δ.In conclusion, the present study demonstrates for the first time that FABP5 enhances the gene expression of SREBP-1c induced by amino acids and hormones for milk fat synthesis in BMECs.