Background: Amniotic fluid-derived stem cells (AFSCs) possess optimal differentiation potential and are a promising resource for cell therapy and tissue engineering. Mouse is a good model to be studied for pre-clinical research. Methods: In this study, we successfully established enhanced green fluorescent protein mouse-derived amniotic fluid stem cells (EGFP-mAFSCs) and investigated whether EGFP-mAFSCs possess the ability to differentiate into cardiomyocytes by in vitro culture.We evaluated stem-cell differentiation using immunofluorescence. Results: This study showed that EGFP-mAFSCs can give rise to spontaneously beating cardiomyocyte-like cells expressing the specific markers c-kit, myosin heavy chain, and cardiac troponin I. Conclusions: We demonstrated that mAFSCs have the in vitro propensity to acquire a cardiomyogenic phenotype and to a certain extent cardiomyocytes; however the process efficiency which gives rise to cardiomyocyte-like cells remains quite low (2 out of 10 were found).