Abstract Body: Unlike the majority of adult mammals, neonatal mice can fully regenerate their hearts with little to no fibrosis, though this regenerative capacity declines over the first week of life. We have previously characterized a cardiomyocyte cluster through snRNA-sequencing that is enriched in mouse hearts during this regenerative stage. These neonatal-enriched cardiomyocytes undergo cell cycle activity following ischemic injury, and differentially express metabolism, stress resistance, and embryonic myocardial markers. However, whether these cardiomyocytes actually contribute to or are required for neonatal regeneration is unknown. Here we show that this cardiomyocyte cluster can also be identified in human neonatal samples, and that its unique cell signature is evolutionarily conserved and differentially upregulated in the cardiomyocytes of known regenerative organisms, such as the zebrafish. Further, we adapted a dual recombinase mouse system driving a TdTomato reporter and the diphtheria toxin receptor to track these neonatal-enriched cardiomyocytes in vivo. Following ischemic damage, we found that these specialized cardiomyocytes are enriched in the border-zone, and ultimately make up a large proportion of the regenerated myocardium. By administering diphtheria toxin, we were able to selectively ablate these cardiomyocytes, finding they are in fact necessary for neonatal regeneration. To translate these in vivo results to clinical relevance, we identified and overexpressed conserved genes that make up the neonatal-enriched cardiomyocyte program, finding novel genetic markers capable of providing protection from ischemic injury in non-regenerative mice. Finally, we identified conserved transcription factors (TFs) upstream of the neonatal-enriched cardiomyocyte program using a multiomic dataset of human neonatal samples. By overexpressing these TFs in iPSC-derived cardiomyocytes, we have identified a “regenerative cocktail” capable of reprogramming human cardiomyocytes to the neonatal, pro-regenerative cardiomyocyte fate. Together, these results uncover an evolutionarily conserved cardiomyocyte cluster necessary for neonatal regeneration which express a pro-regenerative genetic program, providing exciting avenues towards therapeutics for ischemic injury.