Regenerative therapy is a new strategy for the end-stage heart failure; however, the ideal cell source has not yet been established for this therapy. We expected that the amnion might be an ideal cell source for cardiac regenerative therapy and that the differentiation potency of the human amnion mesenchymal cells (hAMCs) could be improved by overexpression of Oct4, a key factor that maintains the undifferentiated state. A plasmid vector was made by insertion of the Oct4 open reading frame (ORF) under control of a cytomegalovirus (CMV) promoter (pCMV-hOct4) and transfected into hAMCs by electroporation. The optimum induction time was investigated by comparing the quantity of stem cell-specific mRNAs, cardiac-specific mRNAs, and cardiac-specific proteins with time. hAMCs already expressed cardiac-specific proteins such as Nkx2.5 and Connexin43. After pCMV-hOct4 transfection, endogenous Oct4 mRNA and other stem cell markers showed a transient increase. With 5-azacytidine treatment, quantities of the cardiac-specific mRNAs, such as GATA4 and myosin light-chain-2v (Mlc-2v), were increased significantly. After Oct4 overexpression, the highest expression of cardiac-specific mRNAs and stem cell makers was seen at almost the same time. Furthermore, more mature myocardial contraction proteins were observed when hAMCs were induced at specific optimal times after gene transfection. In conclusion, hAMCs were activated to an undifferentiated state by overexpression of Oct4, and their cardiac differentiation potency was improved. Thus, the single-time transfection of the Oct4 expression vector may be a useful strategy for effective cell therapy. The use of cryopreserved hAMCs in cell therapy still requires more investigation.