Tanshinone VI (TanVI) is prepared from Salvia miltiorrhiza Bunge, which has been widely used for treatment of cardiovascular disease in Chinese medicine. In our previous study, we showed the differentiation effect of TanVI on cardiosphere-derived cell (CDC) prepared from adult rat cardiac tissue. However, the exact mechanism of TanVI underlying the differentiation from cardiac stem cells to cardiomyocytes is yet unclear. In this study, we examined the effect of TanVI on the intracellular signaling pathway in CDCs during myocardial differentiation. After CDCs in the presence of TanVI were cultured, the cells expressed a cardiomyocyte marker cardiac troponin T. Concomitantly, an increase in the expression of the cardiac transcription factor Nkx2.5 was also observed in TanVI-treated CDCs. Treatment of the cells with TanVI resulted in reduction of GSK3β phosphorylation. The β-catenin expression level in CDCs was also decreased. Furthermore, TanVI reduced the phosphorylation levels of Erk1/2. These results suggest that TanVI enhances the differentiation of CDCs into cardiomyocytes via an attenuation of GSK3β,β-catenin and Erk1/2 phosphorylation.