Objective To investigate the biocharacteristics and myocardium differentiation capacity of human amniotic fluid-derived c-kit+ and c-kit- mesenchymal stem cells in vitro. Methods Fourteen samples of human amniotic fluid were routinely obtained from amniocenteses of second trimester gestation (15-31weeks). C-kit+ amniotic fluid stem (AFS) cells were sorted by flow cytometry and were compared with c-kit- stem cells for the biocharacteristics and adipogenic, osteogenic and myocardium differentiation capacities. Verification and differentiation was evaluated by using RT-PCR and Weston blot. Result Our findings revealed that 16 to 22 weeks of gestation was the optimal duration to obtain c-kit+ stem cells. Following the sorting, c-kit+ cells had similar morphological and proliferative characteristics with the c-kit- AFS cells as demonstrated by growth curves. Both the c-kit+ and c-kit- AFS cells had mesenchymal stem cell characteristics through surface marker identification by flow cytometry and immunocytochemical analysis. They expressed mesenchymal stem cell markers (CD29,CD44,CD73,CD90,CD105) but not hematopoietic stem cells makers(CD34,CD45). The cells were positive for Class I major histocompatibility (MHC) antigens (HLA-ABC), and negative for MHC Class II (HLA-DR). Oct-4 and SSEA-4 expression were stronger in c-kit+ stem cells than in c-kit- stem cells. Both two types of stem cells can differentiate along adipogenic and osteogenic lineages. However, the myocardium differentiation capacity was enhanced in c-kit+ AFS by detecting the GATA-4, cTnT, α-actin, Cx43 mRNA and protein expression through RT-PCR and Western blot analysis after myocardium induction. Only GATA-4, a marker for early stage of immature myocardium like cells, was detected from the myocardium induced c-kit- AFS cells. Conclusion Both c-kit+ and c-kit- amniotic fluid-derived stem cells have mesenchymal stem cells characteristics. They can differentiate along adipogenic and osteogenic lineages. C-kit+ AFS cells may have the potential clinical application for myogenesis in cardiac regenerative therapy.