Three-dimensional in vitro tumor models are needed to obtain more information about drug behavior in tumors. The aim of this study is to establish a new model for hepatocellular carcinoma (HCC) using decellularized rat livers. After generating the rat liver scaffolds, HepG2 liver cancer cells were perfused via the portal vein and placed in a bioreactor for 10 days. Histology was performed to analyze cell distribution within the scaffolds. Function and tumor-related gene expression were examined by polymerase chain reaction (PCR). We evaluated the function of HepG2 cells grown on scaffolds in the presence of a well-known anti-cancer drug to investigate the potential application of our system for drug screening. The scaffolds were devoid of cellular materials and preserved extracellular matrix components. HepG2 cells grew well on the scaffolds. The PCR results showed that the cells maintained function and invasion ability at significantly higher levels than cells grown on two-dimensional (2-D) dishes or spheroids on Matrigel. Unlike the 2-D cultures, albumin secretion and alpha-fetoprotein expression in three-dimensional cultures were less susceptible to lower concentrations of the drug. Cells grown in scaffolds seemed to respond to the drug in an analogous manner to its known activity in vivo. These findings strengthen the potential use of rat liver scaffolds for screening HCC drugs.