Abstract: Hepatocytes are the major parenchymal cells (PC) in the liver and present an important role in liver metabolism. Hepatocytes are considered a gold standard tool for drug toxicity/screening or liver disease modeling. However, the maturation and functions of hepatocytes are lost under routine 2- dimensional (2D) culture conditions. Recent studies revealed that the interactions between hepatocytes and non-parenchyma cells (NPC) under 3D culture conditions can be an alternative option for optimizing hepatocyte maturation. Co-culture of hepatocytes with NPC simplifies the in-vitro liver disease models of fibrosis, steatosis and non-alcoholic fatty liver disease (NAFLD), cholestasis, and viral hepatitis. This review described the co-culture of liver PC with NPC under 2D and 3D culture systems.