Background Vietnamese medicine tried and tested certain bioactive compounds from plants to increase the rate of tissue immunomodulation, regeneration, and differentiation. Although there are many research papers discovered about phytochemicals of Rehmannia glutinosa Libosch and differentiation induction potential of some substances purified from this herbal, it finds difficult to seek research that investigated the effect of hot water-extracted R. glutinosa Libosch (RGE) on proliferation and cardiogenic differentiation of mesenchymal stem cells, even though it has commonly been used for a long time because of its function as a restorative and as a critical role in cardiovascular treatment in traditional. Results Our research indicated that RGE has many predicted bio-pharmacological effects, and the RGE is demonstrated that it is non-toxic to UC-MSCs (IC50 = 1274 ppm). It also stimulates the proliferation and migration of UC-MSCs at various concentrations, especially at the RGE concentration of 50 ppm, during four days of treatment. On the other hand, the RGE can induce the cardiac pre-differentiation process from the fifth day to the fifteenth day after treatment, which was proven through both molecular and cellular (morphology evidence) levels like the up-regulation of GATA4, Nkx2.5, cTnT α-MHC, Desmin genes; the expression of Desmin protein, the appearance of two-nuclei cells, connecting process of adjoining cells, the cytoplasmic striations. Conclusion The RGE could either stimulate proliferation–migration of MSCs or induce the cardiac pre-differentiation process. This extract can be classified as non-toxic to the UC-MSCs.