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Human amniotic fluid stem cells (hAFSCs) derived from second-trimester amniocentesis were evaluated for the therapeutic potential of cardiac repair. Whether hAFSCs can be differentiated into cardiomyogenic cells and toward the maturation of endothelial cell lineage was investigated in vitrousing mimicking differentiation milieu. Employing an immune-suppressed rat model with experimental myocardial infarction, an intramyocardial injection was conducted with a needle directly into the peri-infarct areas. There were three treatment groups: sham, saline, and hAFSCs (n≥ 10). When cultured with rat neonatal cardiomyocytes or in endothelial growth medium-2 enriched with vascular endothelial growth factor, hAFSCs were differentiated into cardiomyocyte-like cells and cells of endothelial lineage, respectively. After 4 weeks, hAFSC-treated animals showed a preservation of the infarcted thickness, an attenuation of left ventricle remodeling, a higher vascular density, and thus an improvement in cardiac function, when compared with the saline injection group. Survival and proliferation of the transplanted hAFSCs were revealed by immunohistochemical staining. Expressions of the cardiac-specific markers such as Nkx2.5, α-actinin, and cardiac Troponin T were observed in the transplanted hAFSCs. Additionally, Cx43 was clearly expressed at the borders of the transplanted/transplanted and host/transplanted cells, an indication of enhancement of cell connection. The results demonstrated that hAFSCs induce angiogenesis, have cardiomyogenic potential, and may be used as a new cell source for cellular cardiomyoplasty.