Adult cardiomyocytes have little ability to regenerate, thus cardiac regeneration therapy represents a potential method for treating severe heart failure. Human amniotic mesenchymal cells (hAMCs) have the potential to be a useful cell source for cardiac regeneration therapy. We attempted to isolate stem cells from hAMCs and differentiate them into cardiomyocytes. Nanog promoter-Cre plasmid and cytomegalovirus (CMV) promoter-loxP-STOP-loxP-Red-puro(r) plasmid were co-transfected into immortalized hAMCs (iHAMs). Nanog-positive iHAMs were treated with 5-azacytidine (5-aza), trichostatin A (TA), activin A (AA), and bone morphogenetic protein-4 (BMP-4), or co-cultured with murine fetal cardiomyocytes for cardiomyocytes differentiation. Isolated Nanog-positive iHAMs were analyzed by quantitative RT-PCR and immunofluorescent staining before and after differentiation. Expression of Nanog, Oct3/4, Sox2, and Klf4 was significantly higher in Nanog-positive than in Nanog-negative iHAMs. Nanog-positive iHAMs were stained for Nanog and Oct3/4 in the nucleus. Nanog-positive iHAMs treated with 5-aza expressed Nkx2.5, GATA-4, human atrial natriuretic peptide (hANP), cardiac troponin T (cTnT), myocin light chain (Mlc)-2a, Mlc-2v, β-myosin heavy chain (β-MHC), hyperpolarization-activated cyclic nucleotide gated channels (HCN)-4, and inwardly rectifying potassium channels (Kir)-2.1. Although Nanog-positive iHAMs treated with TA, AA, or BMP-4 expressed several cardiac markers, no contraction was observed. Co-cultured Nanog-positive iHAMs with murine fetal cardiomyocytes spontaneously contracted in a synchronized manner and expressed the cardiac markers. In conclusion, Nanog-positive hAMCs with characteristics of stem cells were isolated and differentiated into cardiomyocyte-like cells, suggesting that these isolated hAMCs could be a useful cell source for cardiac regeneration therapy.