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Cardiovascular diseases remain the leading cause of morbidity and mortality worldwide; however, effective clinical therapies that achieve myocardial regeneration are still lacking, underscoring the urgent need for novel therapeutic strategies. In this study, we identified an RNA-binding protein Ptbp1, a key component of RNA metabolism, as a critical regulator of cardiomyocyte proliferation via the formation of biomolecular condensates. We found that Ptbp1 exhibits granule-like structures in cardiomyocyte nuclei during fetal to early postnatal stages. These Ptbp1 foci are phase-separated liquid droplets formed via its multivalent RNA binding domains. Functional analyses revealed that Ptbp1 is essential for cardiomyocyte proliferation in neonatal mouse primary culture assays. Furthermore, proximity labeling using a primary antibody-based approach combined with RIP-seq in highly purified cardiomyocyte populations demonstrated that these condensates are enriched with multiple proliferation-associated factors. Our findings identify a novel phase-separated condensate in cardiomyocytes and provide a new potential therapeutic target for cardiac regeneration.