Background: The important role of SMAD6 and several microRNAs (miRNAs), such as miR-17-5p, miR-26b-5p, and miR-32-5p, has been demonstrated in controlling the proliferation and differentiation of cardiomyocytes (CMs). Hence, this study was designed to assess the role of these regulatory factors in cardiac cell generation from human endometrium-derived mesenchymal stem cells (hEMSCs). Methods: To induce transdifferentiation into CMs, hEMSCs were treated with a cardiac-inducing medium containing 5-azacytidine and bFGF for 30 days. Immunofluorescence staining and qRT-PCR, respectively, were used to measure the protein levels of SMAD6 and the mRNA expression of SMAD6 and the three miRNAs every six days. Results: Our findings demonstrated the mesenchymal stem cell properties of hEMSCs and their ability to differentiate into various types of mesenchymal stem cells. The differentiated hEMSCs exhibited morphological features resembling CMs. During the induction period, the number of positive cells for SMAD6 protein and the expression level of miR-26b-5p increased and peaking on days 24 and 30, while the expression levels of miR-17-5p and miR-32-5p decreased. The Pearson correlation coefficients revealed that SMAD6 level is inversely correlated with miR-17-5p and miR-32-5p and directly correlated with miR-26b-5p. Conclusions: Our results indicate that miR-17-5p, miR-26b-5p, miR-32-5p, and SMAD6 are potentially involved in the molecular signaling pathways of transdifferentiation of hEMSCs to CMs.