Mesenchymal stem cells (MSCs) possess the capacity to differentiate into various cells of the myogenic, osteogenic, chondrogenic, and adipogenic lineages. However, a short life span and the low efficacy into cardiomyogenic differentiation of MSCs have limited the potential use of MSCs for cardiac cell therapy. Retinoic acid (RA) plays an important role for cardiac morphogenesis and myocardial differentiation. To develop a high-efficacy cardiac differentiation system in MSCs, we investigated the effect of RA on cardiac differentiation of MSCs derived from mouse bone marrow. MSCs isolated from adult ICR bone marrow were cultured on gelatin-coated dishes for 7 days in vitro to deplete hamatopoietic lineage cells. Homogenous Sca-1+ cells were purified from mixed bone marrow MSCs by a magnetic cell sorting system (MACS), and treated with 10 nM of all-trans RA for 10 or 30 days in vitro. Real-time RT-PCR analysis showed that bone morphogenetic protein (BMP)-2 was remarkably up-regulated in Sca-1+ MSCs treated with RA for 10 days whereas expression of transcription factors which was known to be involved in cardiac differentiation such as BMP-4, Wnt-3a, -5a, -7a, -8a, -11 was not changed. When treated with RA for 30 days, cardiomyocyte-specific markers such as atrial natriuretic factor (ANF), desmin, and cardiac transcription factors GATA-4, Nkx2.5 were expressed in Sca-1+ cells. By immunohistochemistry, cardiac differentiation of RA-treated Sca-1+ cells was further confirmed by detection of cardiac specific markers such as desmin, sarcomeric alpha-actinin, cardiac myosin heavy chain, and ANF, respectively. These results demonstrate that RA can stimulate cardiac differentiation of Sca-1+ mesenchymal stem cells isolated from adult murine bone marrow via the up-regulation of BMP-2 molecule.