Abstract Body: Cardiomyocyte (CM) maturation is a highly orchestrated process essential for perinatal cardiac health, encompassing profound structural remodeling, a metabolic transition from glycolysis to fatty acid oxidation, and mitochondria maturation. Yet, the upstream regulatory mechanisms driving these events remain incompletely understood. We recently established protein neddylation, a covalent attachment of the ubiquitin-like protein NEDD8 to target substrates, as a crucial post-translational modification governing CM maturation, though its specific downstream effectors remain elusive. To address this knowledge gap, we utilized an AAV9-driven perinatal CM-specific knockout (KO) of Nae1 and Nedp1 to demonstrate that restoring non-cullin neddylation fails to rescue Nae1 KO-induced cardiac dysfunction, underscoring a critical dependence on neddylated cullins in the heart. Concurrently, we observed that inhibiting neddylation severely impairs mitochondrial turnover. Furthermore, proximity biotinylation assays identified Cullin-RING ubiquitin ligase 5 (CRL5) localized to CM mitochondria. Consequently, we generated CM-specific Cul5 KO mice via αMHC^Cre. Ablation of Cul5 precipitated cardiac dysfunction as early as one month of age, subsequently progressing to overt heart failure by four months. Both bulk and single-nucleus RNA-sequencing of Cul5-deficient hearts revealed a significant downregulation of oxidative phosphorylation cascades and adult electrophysiological markers, alongside a persistent fetal gene program, indicative of arrested maturation. To isolate primary maturation defects from secondary cardiomyopathic remodeling, we implemented a MyoAAV-driven mosaic Cul5 KO model with maintained cardiac function. Isolation of adult CMs from these hearts revealed pronounced features of immaturity in the KO cells, including decreased cell size, a reduction in characteristic rod-shaped morphology, and compromised mitochondrial turnover. In vitro silencing of Cul5 corroborated these findings, leading to dysregulated maturation gene networks and impaired oxidative respiration. In conclusion, we proposed that CRL5 acts as a requisite ubiquitin ligase orchestrating CM maturation by mediating mitophagy. This axis ensures the clearance of damaged organelles to meet the high energy demands of the perinatal metabolic shift, unveiling a novel regulatory paradigm in cardiac maturation.