Abstract Stem cell-based therapy is a promising intervention for ischemic heart diseases. However, the functional integrity of stem cells is impaired in an ischemic environment. Here, we report a novel finding that heat shock significantly improves Sca-1+stem cell survival in an ischemic environment by the regulation of the triangle: heat shock factor 1 (HSF1), HSF1/miR-34a, and heat shock protein 70 (HSP70). Initially we prove that HSP70 is the key chaperone-mediating cytoprotective effect of heat shock in Sca-1+cells and then we establish miR-34a as a direct repressor of HSP70. We found that HSP70 was downregulated in heat shocked Sca-1+ stem cells (HSSca-1+ cells). Intriguingly, we demonstrate that the downregulation of miR-34a is attributed to HSF1-mediated epigenetic repression through histone H3 Lys27 trimethylation (H3K27me3) on miR-34a promoter. Moreover, we show that heat shock induces exosomal transfer of HSF1 from Sca-1+ cells, which directs ischemic cardiomyocytes toward a prosurvival phenotype by epigenetic repression of miR-34a. In addition, our in vivo study demonstrates that transplantation of HSSca-1+ cells significantly reduces apoptosis, attenuates fibrosis, and improves global heart functions in ischemic myocardium. Hence, our study provides not only novel insights into the effects of heat shock on stem cell survival and paracrine behavior but also may have therapeutic values for stem cell therapy in ischemic heart diseases. Stem Cells 2014;32:462–472