Previous studies have revealed c-kit-positive (c-kit(+)) cardiac stem cells (CSCs) in the adult mammalian heart and these cells could be a suitable cell source for heart regeneration therapy. However, these cells have not been fully evaluated in terms of characterization and effect of long-term culture, which is necessary for their safe and optimal usage. Therefore, we isolated c-kit(+) CSCs from adult rat hearts to characterize these cells and investigate stability over long-term culture. We performed isolations of c-kit(+) CSCs 11 times and passaged them 40 times in a bulk culture system; we termed these cultures, bulk culture CSCs (CSC-BC). c-kit(+) CSCs expressed stemness genes and exhibited stem cell properties of single cell-derived clone formation, cardiosphere generation, and potential to differentiate into the three main cardiac lineages: cardiomyocyte, smooth muscle, and endothelial cells in vitro. Over long-term culture, some CSC-BC up-regulated GATA-4 expression, which resulted in enhanced cardiomyocyte differentiation, suggesting that the GATA-4 high c-kit(+) CSCs have potent cardiac regenerative potential. We also observed the spontaneous differentiation into cells other than cardiac lineages, such as adipocyte and skeletal myocyte. This effect of long-term culture on the c-kit(+) CSCs has not been previously reported. Interestingly, when c-kit(+) CSCs were co-cultured with adult rat cardiomyocytes, we found increased cardiomyocyte survival, and the growth factors, insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), appeared to be responsible factors. The present study suggests that c-kit(+) CSCs have great therapeutic potential yet should be further investigated and optimized as a cell source for regenerative therapies prior to transplantation.