Despite previous studies demonstrating a cardioprotective role of estradiol via its estrogen receptor (ER)alpha, the underlying mechanisms remain unclear. Here we aimed to define ERalpha-involved mechanisms against cardiac injury. Seven days after myocardial infarction in male rats, cardiac ERalpha was upregulated in post-infarct cardiac c-kit+ cells accumulating in periinfarct myocardium as shown by Western blotting and immunofluorescence staining. Further, we isolated post-infarct cardiac c-kit+ cell population by modified magnetic activated cell sorting (MACS) and fluorescence activated cell sorting (FACS), and confirmed predominant ERalpha expression in this post-infarct cardiac c-kit+ cell population by real-time PCR. These post-infarct cardiac c-kit+ cells, characterized by upregulated transcription factors implicated in cardiogenic differentiation (GATA-4, Notch-2) and genes required for self-renewal (Tbx3, Akt), maintained a stable phenotype in vitro for more than 3 months. ERalpha stimulation supported proliferation but prevented differentiation of undifferentiated myoblast cells. When adult myocytes isolated from infarcted rat hearts were co-cultured with post-infarct cardiac c-kit+ cells, ERalpha stimulation inhibited apoptosis and enhanced survival of these myocytes. These findings suggest that cardiac ERalpha supports survival of cardiomyocytes through post-infarct cardiac c-kit+ cells, which may contribute to cardioprotection against cardiac injury.