© 2019 Elsevier Inc. Cardiomyocytes undergo dramatic changes during the fetal to neonatal transition stage to adapt to the new environment. The molecular and genetic mechanisms regulating these changes remain elusive. In this study, we showed Sema6D as a novel signaling molecule regulating perinatal cardiomyocyte proliferation and maturation. SEMA6D is a member of the Semaphorin family of signaling molecules. To reveal its function during cardiogenesis, we specifically inactivated Sema6D in embryonic cardiomyocytes using a conditional gene deletion approach. All mutant animals showed hypoplastic myocardial walls in neonatal hearts due to reduced cell proliferation. We further revealed that expression of MYCN and its downstream cell cycle regulators is impaired in late fetal hearts in which Sema6D is deleted, suggesting that SEMA6D acts through MYCN to regulate cardiomyocyte proliferation. In early postnatal mutant hearts, expression of adult forms of sarcomeric proteins is increased, while expression of embryonic forms is decreased. These data collectively suggest that SEMA6D is required to maintain late fetal/early neonatal cardiomyocytes at a proliferative and less mature status. Deletion of Sema6D in cardiomyocytes led to reduced proliferation and accelerated maturation. We further examined the consequence of these defects through echocardiographic analysis. Embryonic heart deletion of Sema6D significantly impaired the cardiac contraction of male adult hearts, while having a minor effect on female mutant hearts, suggesting that the effect of Sema6D-deletion in adult hearts is sex dependent.