ALKBH5 regulates cardiomyocyte proliferation and heart regeneration by demethylating the mRNA of YTHDF1

N⁶-methyladenosine (m⁶A) RNA modification, a dynamic and reversible process, is essential for tissue development and pathogenesis. However, the potential involvement of m⁶A in the regulation of cardiomyocyte (CM) proliferation and cardiac regeneration remains unclear. In this study, we aimed to investigate the essential role of m⁶A modification in heart regeneration during postnatal and adult injury. <b>Methods and results:</b> In this study, we identified the downregulation of m⁶A demethylase ALKBH5, an m6A "eraser" that is responsible for increased m⁶A methylation, in the heart after birth. Notably, <i>ALKBH5</i> knockout mice exhibited decreased cardiac regenerative ability and heart function after neonatal apex resection. Conversely, forced expression of ALKBH5 via adeno-associated virus-9 (AAV9) delivery markedly reduced the infarct size, restored cardiac function and promoted CM proliferation after myocardial infarction in juvenile (7 days old) and adult (8-weeks old) mice. Mechanistically, ALKBH5-mediated m⁶A demethylation improved the mRNA stability of YTH N⁶-methyladenosine RNA-binding protein 1 (YTHDF1), thereby increasing its expression, which consequently promoted the translation of Yes-associated protein (YAP). The modulation of ALKBH5 and YTHDF1 expression in human induced pluripotent stem cell-derived cardiomyocytes consistently yielded similar results. <b>Conclusion:</b> Taken together, our findings highlight the vital role of the ALKBH5-m⁶A-YTHDF1-YAP axis in the regulation of CMs to re-enter the cell cycle. This finding suggests a novel potential therapeutic strategy for cardiac regeneration.