Adult heart suffering from increased workload will undergo myocardial hypertrophy, subsequent cardiomyocyte (CM) death, and eventually heart failure. However, the effect of increasing afterload on the neonatal heart remains unknown. We performed ascending aortic constriction (AAC) in neonatal rats 8-12 hours after birth (P0, P indicates postpartum). Seven days after surgery, in vivo heart function was evaluated using cardiac ultrasonography. Haematoxylineosin and Masson staining were used to assess CM diameter and collagen deposition. Moreover, expression of both EdU and Ki67 were evaluated to determine DNA synthesis levels, and pH3 and aurora B as markers for mitosis in CMs. CM isolation was performed by heart perfusion at P0, P3, P5, and P7, respectively. CM number on P0 was 1.01 ± 0.29 × 106. We found that CM cell cycle activation was significantly increased among constricted hearts, as demonstrated by increased Ki67, EdU, pH3, and aurora B positive cells/1000 CMs. At day 7 (P7), constriction group hearts manifested increased wall thickness (0.55 ± 0.05 mm versus 0.85 ± 0.10 mm, P < 0.01, n = 6), and improved hemodynamics as well as left ventricular ejection fraction (65.5 ± 3.7% versus 77.7 ± 4.8%, P < 0.01, n = 6). Of note, the population of CMs was also markedly increased in the constriction group (2.92 ± 0.27 × 106 versus 3.41 ± 0.40 × 106, P < 0.05, n = 6). In summary, we found that during the first week after birth significant numbers of neonatal CMs can reenter the cell cycle. Ascending aortic constriction promotes neonatal rat CM proliferation resulting in 16.7% more CMs in the heart.