Nonalcoholic fatty liver disease is a chronic inflammatory liver disease. It is associated with obesity and type 2 diabetes. Oxycholesterols are metabolites of cholesterol, and several of them can act on the G protein-coupled receptor, G protein-coupled receptor 183 (GPR183)/Epstein-Barr virus-induced gene 2. We found expression of GPR183 in human hepatoma cell lines and in vivo induction of GPR183 expression in mouse livers after high-fat diet feeding. Therefore, the role of oxycholesterols and GPR183 in hepatocytes was studied using a model of hepatic steatosis induced by liver X receptor (LXR) activation. LXR activation by T0901317 resulted in fat accumulation in Hep3B human hepatoma cells. This lipid accumulation was inhibited by 7α,25-dihydroxycholesterol, the most potent agonist of GPR183. The protective effects of 7α,25-dihydroxycholesterol were suppressed by a specific GPR183 antagonist, NIBR189 [(2E)-3-(4-Bromophenyl)-1-[4-4-methoxybenzoyl)-1-piperazinyl]-2-propene-1-one]. T0901317 treatment induced expression of the major transcription factor for lipogenesis, sterol regulatory element-binding protein 1c (SREBP-1c). 7α,25-Dihydroxycholesterol inhibited the induction of SREBP-1c proteins in a GPR183-dependent manner. Using inhibitors specific for intracellular signaling molecules, 7α,25-dihydroxycholesterol-induced suppression of hepatocellular steatosis was shown to be mediated through G_i/o proteins, p38 mitogen-activated protein kinases, phosphoinositide 3-kinase, and AMP-activated protein kinase. In addition, the inhibitory effect of 7α,25-dihydroxycholesterol was validated in HepG2 cells and primary mouse hepatocytes. Therefore, the present report suggests that 7α,25-dihydroxycholesterol-GPR183 signaling may suppress hepatocellular steatosis in the liver. SIGNIFICANCE STATEMENT: Oxycholesterols, which are metabolites of cholesterol, act on the G protein-coupled receptor, G protein-coupled receptor 183 (GPR183)/Epstein-Barr virus-induced gene 2, which is expressed in human hepatoma cell lines, and its expression is induced in vivo in mouse livers after high-fat diet feeding. Activation of GPR183 inhibits fat accumulation in primary mouse hepatocytes and HepG2 cells through G_i/o proteins, p38 mitogen-activated protein kinases, phosphoinositide 3-kinase, and AMP-activated protein kinase.