Mesenchymal stem cells (MSCs) can differentiate into different types of cells and thus have tremendous potential for cell therapy and tissue engineering. 5-Azacytidine (5-aza), a DNA demethylation reagent, has been reported to induce MSCs to differentiate into cardiomyocytes in vitro. To determine a global effect of 5-aza on MSCs, we investigated the protein expressions of rat MSCs with two-dimensional gel electrophoresis (2-DE). We have generated a proteome reference map of rat MSCs, and have successfully identified 34 proteins with MALDI-TOF-MS analysis. Nine proteins showed distinct regulation in MSCs after 5-aza treatment. The proteins regulated by 5-aza included cytoskeletal proteins, cadmium-binding proteins, and metabolic proteins, etc. These proteins have been reported to be involved in cell proliferation and differentiation through different signaling pathways, and the molecular mechanism of MSCs differentiation is discussed at the proteome level.