Abstract Body: Deubiquitinases (DUBs) regulate protein stability and various cellular processes by removing ubiquitin from substrates. OTUD6B, a member of the OTU DUB family, has been linked to intellectual disabilities and congenital heart defects through compound heterozygous or homozygous variants in the OTUD6B gene. However, its molecular and pathophysiological roles in the heart remain poorly understood. Here, we demonstrate that OTUD6B is essential for ventricular chamber development and postnatal survival. OTUD6B is ubiquitously expressed in mouse embryos. Hypomorphic Otud6b^neo/neo mice, with a 90% reduction in OTUD6B protein, were viable, fertile, and showed no gross abnormalities up to 24 weeks. In contrast, Otud6b^-/- mice, with biallelic loss of OTUD6B, exhibited growth retardation and failed to survive beyond postnatal day 1 (P1). Histological analysis revealed significant cardiac defects in Otud6b^-/-, including ventricular septal defects (VSD) and severe bi-ventricular wall thinning as early as embryonic day 16.5 (E16.5). Echocardiography at E20.5 indicated signs of heart failure. Transcriptomic analysis of E14.5 hearts revealed perturbations in cell cycle regulation, fatty acid metabolism, myogenesis and cardiomyocyte maturation in Otud6b^-/-. The severe cardiac phenotype arises from reduced cardiomyocyte proliferation due to defective cell cycle progression, characterized by S-phase arrest and decreased binucleation in OTUD6B-deficient cardiomyocytes. Moreover, homozygous Otud6b knockin mice (Otud6b^KI/KI) bearing pathogenic variants R145* in Otud6b gene recapitulated the myocardial hypoplasia, ventricular septal defects, and perinatal lethality observed in Otud6b^-/-, mirroring cardiac defects seen in patients with R145* mutations. Taken together, our findings establish an essential role for OTUD6B in perinatal ventricular chamber development, likely by fine-tuning cell cycle regulators during heart development.