Fluoxetine is a first-line selective serotonin reuptake inhibitor widely applied for the treatment of depression; however, it induces abnormal hepatic lipid metabolism. Considering decreased expression or function of glucose-6-phosphatase (G6Pase), a key enzyme in gluconeogenesis, or the upregulation of fatty acid uptake, causes hepatic lipid accumulation. The aim of this study was to elucidate whether G6Pase regulation and fatty acid uptake alteration contribute to fluoxetine-induced abnormal hepatic lipid metabolism. Our study revealed that 8-week oral administration of fluoxetine dose-dependently increased hepatic triglyceride, causing hepatic steatosis. Concomitantly, the expression of G6Pase in mouse livers and primary mouse hepatocytes (PMHs) was downregulated in a concentration-dependent manner. Furthermore, fluoxetine increased the concentrations of glucose-6-phosphate (G6Pase substrate) and acetyl CoA (the substrate for de novo lipogenesis) in mouse livers. Additionally, fluoxetine also induced lipid accumulation and downregulated G6Pase expression in HepG2 cells. However, the uptake of green fluorescent fatty acid (BODIPY™ FL C16) in PMHs was not changed after fluoxetine treatment, indicating that fluoxetine-induced hepatic steatosis was not associated with fatty acid uptake alteration. In conclusion, fluoxetine downregulated hepatic G6Pase expression, subsequently enhanced the transformation of glucose to lipid, and ultimately resulted in hepatic steatosis, but with no impact on fatty acid uptake.