Myocardial infarction (MI) causes irreversible loss of cardiomyocytes and can lead to heart failure. Previous studies have shown that hexokinase 2 (HK2) is significantly upregulated during the proliferative stage of cardiac injury. However, its role and mechanism in myocardial regeneration have not been clarified. In this study, we aimed to explore the mechanism of HK2 in promoting myocardial regeneration. The results indicated that the expression level of HK2 was significantly upregulated in the heart tissue of neonatal mice. In addition, HK2 overexpression significantly increased the positive rate of EdU, pH3, and Ki67 in cardiomyocytes. Moreover, HK2 overexpression still improved the index of proliferation potential of cardiomyocytes following inhibition of the glycolytic pathway by 2-DG. In vivo, HK2 overexpression promoted myocardial regeneration, reduced fibrosis, and improved cardiac function in neonatal mice following apical resection. Mechanistically, we found that MYH9 was the target of HK2, and HK2 promoted cardiomyocyte proliferation by interacting with MYH9 and regulating the GSK3 beta/beta-catenin signaling pathway. Finally, the results demonstrated that HK2 improved cardiac function, inhibited myocardial infarction fibrosis, and promoted cardiomyocyte proliferation in adult mice model of myocardial infarction. In conclusion, we show that HK2 can regulate the cell cycle, promote cardiomyocyte proliferation, and improve myocardial infarction injury by targeting MYH9 via the GSK3 beta/beta-catenin signaling pathway. This study presents a new target for promoting myocardial regeneration therapy after infarction.