The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene program that promotes cell division within this population is not fully understood. Here, we demonstrate increased EdU incorporation in cardiomyocytes at 3 days post-myocardial infarction (MI) in mice. By applying multi-color lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the borderzone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing (scRNA-seq) data from cardiomyocytes at 3 days post ischemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes (NRCM) and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is key for regeneration to occur.