Contents: Previously, we have demonstrated that chronic alcohol consumption promotes the development of metabolic syndrome via the impaired glucose and/or insulin homeostasis. However, the exact molecular mechanisms and its role involved in chronic ethanol consumption-induced the impairment of glucose homeostasis are not fully understood. Here, we know that serum levels of free fatty acid (FFA) and ceramide are significantly increased in chronic ethanol-fed mice model, correlated with the increase of hepatic steatosis, serum or hepatic triglyceride and TNF-a, which are remarkably attenuated in TNFR1-/-mice. In vitro HepG2 or primary hepatocytes, treated ethanol or palmitic acid highly induces lipid accumulation and the reduction of beta-oxidations, which were reversely attenuated by the pretreatment of myriocin, an inhibitor of ceramide production from FFA, suggesting the essential role of ceramide in ethanol or palmitic acid-mediated hepatic steatosis. Interestingly, we found that a stress inducible-transcription factor ATF3 may play as a novel target molecule involved in the regulation of hepatic steatosis by ethanol-FFA-ceramide axis. Ethanol- or palmitic acid-induced ATF3, which is also regulated by TNFR1-dependent pathway, is involved in the dysregulation of autophagy via excessive lipid accumulation. Ceramide-induced ATF3 inhibits the flux of autophagy, which is initiated by the formation of lipid vesicles together with oxidative stress and ER stress, and thus highly accumulates autophagosome in the cytoplasm resulting in hepatocytes apoptosis. Taken together, our studies suggest that hepatic steatosis induced in ethanol-fed mice is associated with the formation of ethanol-FFA-ceramide axis. And also, ATF3, considered as their target molecule, may play as a negative regulator in dynamic regulation of autophagy, thus it may trigger the lipotoxicity of hepatocytes via the autophagosome accumulation.