Non-alcoholic fatty liver disease (NAFLD) held a high global prevalence in recent decades. Hepatic lipid deposition is the major characteristic of NAFLD. We aim to explore the mechanisms of psoralen on lipid deposition in NAFLD. The effects of psoralen on insulin resistance, lipid deposition, the expression and membrane translocation of glucose transporter type 4 (GLUT4), autophagy, and lipogenesis enzymes were determined on sodium oleate-induced L02 cells. Chloroquine and 3-MA were employed. The AMP-activated protein kinase alpha (AMPK alpha) was knocked down by siRNA. Psoralen alleviated insulin resistance in sodium oleate-induced L02 hepatocytes by upregulating the expression and membrane translocation of GLUT4. Psoralen inhibited lipid accumulation by decreasing the expression of key lipogenesis enzymes. Psoralen promotes autophagy and the autophagic flux to enhance lipolysis. Psoralen promoted the fusion of the autophagosome with the lysosome. Both chloroquine and 3-MA blocked the effects of psoralen on autophagy and lipid accumulation. The AMPK alpha deficiency attenuated the effects of psoralen on autophagy and lipid accumulation. Our study demonstrated that as an antioxidant, psoralen attenuates NAFLD by alleviating insulin resistance and promoting autophagy via AMPK, suggesting psoralen to be a promising candidate for NAFLD.