Endogenous CSCs are one of the promising cell sources due to self renewal capacity and inherent differentiation potential into cardiomyocytes (CMCs), smooth muscle cells (SMCs), and endothelial cell (ECs). Nestin was upregulated in hearts with end-stage heart failure and expressed in regenerating and migrating cardiac cells. Here, we report nestin+ CSCs isolated from adult murine hearts using hydrogel-based 3-dimensional organ culture and determined their biologic properties. Initially, nestin+ CSCs consistently coexpressed with stem cell marker (Sca-1) as well as cardiac-specific transcription factors (Nkx2.5, Gata-4, Mef2c), but did not express any structural gene. These cells were clonogenic and possessed multipotent differentiation potential into CMCs, ECs, and adipocytes in vitro. Given intravenously after ischemia or doxorubicin treatment, CSCs can traverse the blood vessel barrier, home to injured myocardium, and regenerate injured myocardium. The directly injected CSCs via intramyocardium attenuated fibrosis and thinning of left ventricle at 4 weeks after myocardial infarction. In addition, locally injected CSCs increased blood perfusion rate and microvascular density in ischemic hindlimb mice. This study demonstrates that endogenous nestin+ CSCs have an intrinsic ability to differentiate into CMCs and ECs and could be used as promising candidate for cardiac regeneration.