The cardiosphere-derived cell (CDC) is one of the candidate cells used for cardiac regenerative therapy. Cardiospheres are mixture of cells including c-Kit⁺ cells, Sca-1⁺ cells, and other types of cardiac progenitor cells prepared from adult cardiac tissue. Tanshinone VI (TanVI) is prepared from Salvia miltiorrhiza Bunge, which has been widely used to treat cardiovascular disease in Chines medicine. However, exact mechanisms underlying therapeutic effects for cardiovascular disease are still unclear. In this study, we examined the effect of TanVI on the ability of the CDC to proliferate and differentiate into cardiomyocytes. Treatment of CDCs with TanVI inhibited the proliferation in a concentration-dependent manner. After two weeks of treatment of the cells with TanVI, cells expressing a cardiomyocyte marker cardiac troponin T were clearly observed. Concomitantly, an increase in the expression of the cardiac transcription factor Nkx2.5 was observed in TanVI-treated cells. These results suggest that the promotion of the differentiation from endogenous cardiac progenitor cells into cardiomyocytes is one of the therapeutic mechanisms of TanVI on cardiovascular disease.