Background: Heart failure (HF) is one of the major serious diseases to do harm to human health. Drugs, interventional treatment and heart transplantation are currently the main methods to treat HF. However, they fail to improve the patient's condition. Mesenchymal stem cells (MSCs) can regenerate functional cardiomyocytes and are promising to become a new therapeutic measure to treat heart failure. We assumed that Rehmannia glutinosa oligosaccharide (RGOs) has the synergistic effect with Nkx2.5 transfected MSCs (Nkx2.5) transplantation in treatment heart failure. Methods: MSCs and Nkx2.5 were preconditioned by RGOs. The apoptosis rate was detected by flow cytometry and the expressions of cardiac specific genes were analyzed with quantitative real-time PCR and Western blot in vitro. Heart failure models were duplicated by injecting doxorubicin (total dose of 15mg/kg) intraperitoneally in male SD rats. When the models were prepared, rats were randomly divided into 6 groups: Control (CON), HF, MSCs, Nkx2.5, RGOs and RGOs combined with Nkx2.5 (RGOs+Nkx2.5) group. Echocardiography was used to detect cardiac function in rats. HE staining was used to observe the pathological changes of myocardium, and Masson staining was used to calculate the collagen volume fraction to detect the degree of myocardial fibrosis. The total mRNA was extracted to detect the following genes including cTnI, CX43, TGF-β1, Collagen I, MEF2 and GATA4 by Q-PCR. Protein of myocardial tissue was extracted to detect the expression of cTnI, CX43, MEF2 and GATA4, by western blot. Results: RGOs could not enhance cardiac specific gene expressions including CK, α-MHC, however it improved the survival of Nkx2.5 induced by H2O2 in vitro. In rat heart failure models, RGOs alone improved the heart pumping function and decreased collagen volume fraction (CVF), TGF-β1 and collagen I expression, and increased MEF2 and GATA4 mRNA expression. Moreover, RGOs cooperated with Nkx2.5 in improving left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV). Furthermore, RGOs and Nkx2.5 combination also increased CX43 expression, whereas decreased CVF and collagen I expression. Conclusion: RGOs has the synergistic effect with Nkx2.5 gene transfected MSCs transplantation in treatment with heart failure through decreasing myocardial fibrosis, inhibiting ventricular remodeling, and increasing the expressions of GATA4, MEF2.