Objective: To test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/beta-catenin signaling pathway. Methods: Rat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/beta-catenin signaling pathway was evaluated by measuring the phosphorylation status of beta-catenin. Results: Over-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated beta-catenin in the wnt/beta-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/beta-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes. Conclusion: Over-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/beta-catenin signal pathway. (C) 2012 Elsevier Inc. All rights reserved.