Objective To explore experimental study of Jiawei Danshen Yin on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into myocardium-like cells in vitro. Methods Rat BMSCs were isolated by adherent method, and their surface antigen were detected by flow cytometry. The maximal non-toxic concentration of Jiawei Danshen Yin was measured by MTT method. The isolated and purified BMSCs were divided into four groups:normal control group, Jiawei Danshen Yin group, 5-azacitidine group, Jiawei Danshen Yin plus 5-azacitidine group. The expression of desmin, α-myosin heavy chain (α-MHC) and cardiac troponin T (cTnT) protein was detected by western blot. The expression of GATA-4, gap junction connexin 43 (CX43) and Nkx2-5 mRNA was detected by RT-PCR. Results The percentage of CD90 positive cells in the rat BMSCs was higher than 95%, and the proportions of CD34 positive and CD45 positive cells was lower than 5%. The maximum toxic concentration of Jiawei Danshen Yin on BMSCs was 0.625 g/L. Compared with the normal control group, the expression of desmin, α-MHC and cardiac cTnT protein was up-regulated, the expression of GATA-4, CX43 and Nkx2-5 mRNA was up-regulated in Jiawei Danshen Yin group, 5-azacitidine group and Jiawei Danshen Yin plus 5-azacitidine group. The difference was statistically significant (P<0.01). Compared with Danshen Yin group or 5-azacitidine group, the expression of desmin, α-MHC and cardiac cTnT protein was up-regulated, the expression of GATA-4, CX43 and Nkx2-5 mRNA was up-regulated in Jiawei Danshen Yin plus 5-azacitidine group. The difference was statistically significant (P<0.01). Conclusion Jiawei Danshen Yin could induce BMSCs differentiation into myocardium-like cells, which had a synergistic effect with 5-azacitidine.