Heart failure is a life-threatening disease with a poor prognosis. The most common cause of heart failure is myocardial infarction, in which a large number of cardiomyocytes are lost by occlusion of coronary blood flow. In adult mammals, injured hearts cannot regenerate themselves because of the lack of proliferative capacity in cardiomyocytes. In mice, cardiomyocytes permanently exit the cell cycle within 2 weeks after birth. Therefore, understanding what molecular mechanisms underlying this permanent cell cycle arrest would provide an important clue to identify potential therapeutic targets to induce heart regeneration. By integrated analysis of metabolome and transcriptome, we found a drastic increase in nucleotide metabolism in the mouse heart during this postnatal cell cycle withdrawal. Pharmacological inhibition of nucleotide metabolism in neonatal mice extended postnatal cardiomyocyte proliferation window with reduced oxidative DNA damage. Our findings suggest that the nucleotide metabolism is a novel regulator of postnatal cardiomyocyte cell cycle.