Hepatocytes, the major target of hepatitis C virus (HCV), are highly polarized. HCV infection requires extensive trafficking to distinct subcellular domains in the polarized hepatocyte. Polarized cells and three-dimensional organoids are commonly used to study liver functions and differentiation. Researchers have begun adapting these cell culture models that morphologically and physiologically resemble hepatocytes in vivo to study HCV infection. This review summarizes the use of three-dimensional cell culture systems in studies of HCV infection.