Heart disease, especially myocardial infarction (MI), has become the leading cause of death all over the world, especially since the myocardium lacks the ability to regenerate after infarction. The capability of mesenchymal stem cells (MSCs) to differentiate into the cardiac lineage holds great potential in regenerative medicine for MI treatment. In this study, we investigated the potential of human MSCs (hMSCs) to differentiate into cardiomyogenic cell lineages, using 5-azacytidine (5-aza) on electrospun poly(e-caprolactone)-gelatin (PCL-gelatin) nanofibrous scaffolds. Immunofluorescence staining analysis showed that after 15 days of in vitro culture the hMSCs differentiated to cardiomyogenic cells on PCL-gelatin (PG) nanofibers and expressed a higher level of cardiac-specific proteins, such as a-actinin and troponin-T, compared to the MSC-differentiated CMs on tissue culture plates (control). To further induce the cardiac differentiation, vascular endothelial growth factor (VEGF) was incorporated into the nanofibers by blending or co-axial electrospinning, and in vitro release study showed that the growth factor could cause sustained release of VEGF from the nanofibers for a period of up to 21 days. The incorporation of VEGF within the nanofibers improved the proliferation of MSCs and, more importantly, enhanced the expression of cardiac-specific proteins on PG-VEGF nanofibers. Our study demonstrated that the electrospun PG nanofibers encapsulated with VEGF have the ability to promote cardiac differentiation of hMSCs, and might be promising scaffolds for myocardial regeneration