Fluoxetine (FLX), a typical drug belonging to the category of selective serotonin reuptake inhibitors (SSRI), is the most widely prescribed psychoactive drug in the treatment of depression and other mood disorders. It has been demonstrated that the administration of FLX increases the possibility of weight gain and dyslipidemia. We find previously that dysregulation of lipogenic and lipolytic genes is critical in FLX-promoted hepatic lipid accumulation. Therefore, a chronic mild stress depression model and cultured primary mouse hepatocytes were used to investigate the effects and underlying mechanisms of FLX on the promoted hepatic lipid accumulation. The evidence have shown that FLX increases the concentrations of triglyceride (TG) and total cholesterol (TC) in the liver tissues of depressive mice, while only increases TG in the liver tissues of normal mice. FLX induces lipid accumulation in both normal and depressive mice by upregulating the lipogeneic genes Acetyl-CoA carboxylase 1 (ACC1) and Fatty acid synthase (FAS) expression and downregulating the lipolytic genes carboxylesterase 1 (CES1) and CES2. Using primary cultured mouse hepatocytes, it is shown that FLX promotes the expression of transcription factor SREBP1c as well as its proteolytic cleavage and nuclear translocation. FLX significantly suppresses SREBP1c proteolytic cleavage in hepatocytes after the incubation lasting as short as 3 hours, which is a more prompt way than the elevated expression of SREBP1c. Further experiments show that the specific inhibitors of proteases S1P and S2P attenuate FLX-induced SREBP1c activation and hepatic lipid accumulation. As SCAP (SREBP cleavage-activating protein) escorts SREBPs from the endoplasmic reticulum to the Golgi complex where proteases cleave SREBPs and therefore is required for SREBP activation, we find that FLX promotes the protein level of SCAP in a concentration- and time-dependent manner. In conclusion, FLX directly acts on the hepatocytes by facilitating the expression and proteolytic activation of SREBP1c to promote hepatic lipid accumulation. The findings not only provide new insight into the understanding of the mechanisms for SSRI-mediated dyslipidemia effects, but also suggest a novel therapeutic target to interfere.