Fatty liver can progress into serious conditions, and the number of patients with fatty liver disease has risen globally in recent years. Various lipid metabolism disorders can cause fatty liver, and in vitro models, such as hepatoma cell lines, have been utilized in research related to lipid metabolism disorders, including the development of treatment strategies. We previously demonstrated that fresh hepatocytes (PXB-cells^®) from chimeric mice with humanized livers display lipid metabolism similar to that of normal human hepatocytes. Additionally, we developed PXB-cells Lipid Analysis (PXB-cells LA) as a model of non-alcoholic fatty liver disease (NAFLD). PXB-cells LA exhibited increased levels of intracellular lipid droplets and lipids, especially triglycerides, compared to PXB-cells. Additionally, albumin secretion, drug metabolism, bile excretion transporters, mitochondria-derived oxidative phosphorylation, and intracellular adenosine triphosphate levels were attenuated in PXB-cells LA, while inflammatory marker levels were elevated. Collectively, these findings indicate hepatic dysfunction. Additionally, PXB-cells LA showed a fractional profile with a peak in very low-density lipoproteins, similar to PXB-cells. PXB-cells LA also secreted lipoproteins with a higher triglyceride content, associated with NAFLD. Taken together, these results suggest that PXB-cells LA is a useful cellular model of human NAFLD.