Protein arginine methyltransferase 5 (PRMT5) regulates gene expression either transcriptionallyly by symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3, or at the post-translational level by methylation of non-histone target proteins. While emerging evidence suggests that PRMT5 functions as an oncogene, its role in metabolic diseases is not well defined. We investigated the role of PRMT5 in promoting high fat-induced hepatic steatosis. High fat diet up-regulated PRMT5 levels in the liver, but not in other metabolically relevant tissues such as skeletal muscle or white and brown adipose tissue. This was associated with repression of master transcription regulators involved in mitochondrial biogenesis. In contrast, lentiviral shRNA-mediated reduction of PRMT5 significantly decreased PI3K/AKT signaling in mouse AML12 liver cells. PRMT5 knockdown or knockout decreased basal AKT phosphorylation, but boosted the expression of PPARalpha and PGC-1alpha with a concomitant increase of mitochondrial biogenesis. Moreover, by overexpressing an exogenous wild-type or enzyme-dead mutant PRMT5, or by inhibiting PRMT5 enzymatic activity with a small molecule inhibitor, we demonstrated that the enzymatic activity of PRMT5 is required for regulation of PPARalpha and PGC-1alpha expression and mitochondrial biogenesis. Our results suggest that targeting PRMT5 may have therapeutic potential for treatment of fatty liver. Biology, Inc.