Hepatocytes derived from mesenchymal stem cells (MSCs) hold great potential for cell-based therapies for liver diseases. The cell-based therapies are critically dependent on the hepatic differentiation of the MSCs with a high efficiency and on a considerable scale. Recent results have shown that decellularized organs provide a three-dimensional extracellular matrix for the lineage restriction of stem cell maturation. In this study, we compared the cell proliferation and hepatic differentiation of murine MSCs in a biomatrix scaffold from rat liver and in the presence and absence growth factors (GF) with a two-dimensional substrate. In the absence or presence of GF, the dynamic cultured scaffold (DCS) stimulated the MSCs to express endodermal and hepatocyte-specific genes and proteins associated with improved functions, and the cells exhibited the ultrastructural characteristics of mature hepatocytes. When transplanted into CCl₄-injured mice, the cells pretreated with a combination of the DCS and GF exhibited increased survival, liver function, engraftment into the host liver and further hepatic differentiation. The paracrine effect of the transplanted cells on hepatic stellate cells and native hepatocytes played a key role in the treatment of the liver pathology. These studies define an effective method that facilitates the hepatic differentiation of MSCs exhibiting extensive functions and support further research into the use of a decellularized liver matrix as a bioscaffold for liver tissue engineering.