Bone marrow mesenchymal stem cells (BMMSCs) are used in cardiac tissue engineering for the regeneration of diseased hearts. We examined the differentiation of rat BMMSCs into cardiomyocyte-like cells when induced with a combined low dose treatment of transforming growth factor-beta 1 (TGF-beta 1) and 5-azacytidine (5-AZA). Results showed that cell proliferation in the combined low dose treatment group of TGF-beta 1 and 5-AZA was increased compared with the TGF-beta 1 group or the 5-AZA group. The cell apoptosis was relieved by combined TGF-beta 1 and 5-AZA treatment compared to 5-AZA treatment alone. The number of cells positive for myosin heavy chain, connexin-43, alpha-actin, and troponin I in the combined treatment group was higher than those observed in the TGF-beta 1 group or the 5-AZA group. Moreover, the combined low dose treatment group of TGF-beta 1 and 5-AZA reveals the strongest expression of troponin I, alpha-actin, and phosphorylated extracellular signal-regulated protein kinases 1 and 2 (p-ErK1/2) among the treatment groups. These results suggest that the combined low dose treatment of TGF-beta 1 and 5-AZA can improve the differentiation potential of rat BMMSCs into cardiomyocyte-like cells and alleviate cell damage effects in vitro. The mechanism that is involved in influencing differentiation may be associated with p-ErK1/2.