Background: Cardiomyocyte (CM) proliferation decreases after birth but the underlying mechanism is poorly understood. Chromobox 7 (CBX7) regulates the cell cycle but its role in CM proliferation is unknown. Methods: We profiled CBX7 expression in the mouse hearts via qRT-PCR, western blotting, and immunohistochemistry. We knocked down CBX7 by using constitutive and inducible conditional knockout mice (Tnnt2-cre;Cbx7 fl/+ and Myh6-MCM;Cbx7 fl/fl , respectively). We then measured CM proliferation by immunostaining. To examine the role of CBX7 in cardiac regeneration, we employed neonatal cardiac apical resection and adult myocardial infarction (MI) models. We examined the mechanism of CBX7-mediated repression of CM proliferation via co-immunoprecipitation, mass spectrometry, and other molecular techniques. Results: The mRNA expression of Cbx7 was increased at the perinatal stage and sustained in the postnatal heart. The haplodeficiency of Cbx7 (Tnnt2-Cre;Cbx7 fl/+ ) promoted proliferation of neonatal CMs in vivo , leading to increased myocardial wall thickness. Genetic deletion of Cbx7 in CMs ( Cbx7 iCKO, Myh6-MCM;Cbx7 fl/fl ) at P0 resulted in increased CM proliferation (% of Ki67 + CMs: vehicle, 9.5 vs. tamoxifen, 18.3, P < 0.01). In response to cardiac apical resection surgery at P7, Cbx7 iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 44.1 vs. tamoxifen, 54.7, P < 0.05) and less fibrosis at P28 compared to the control. Following the MI surgery, adult (5-months-old) Cbx7 iCKO mice exhibited better cardiac function (% of LVEF: vehicle, 41.4 vs. tamoxifen, 51.5, P < 0.05) and less fibrosis at one month post-MI compared to the control. Mechanistically, CBX7 interacted with TARDBP and positively regulated its downstream target, RBM38, an inducer of the cell cycle arrest, in a TARDBP-dependent manner. Rbm38 was perinatally upregulated in the mouse hearts and overexpression of Rbm38 reduced proliferation of neonatal CMs. Conclusions: CBX7 expression is perinatally increased and inhibits proliferation of CMs by controlling TARDBP/Rbm38 pathway. Genetic ablation of Cbx7 promoted regeneration of neonatal and adult hearts. This is the first study to uncover the role of CBX7 in regulation of CM proliferation and cardiac regeneration.