Abstract Body: Ufmylation is a conserved protein post-translational modification that involves the covalent conjugation of the ubiquitin-like modifier UFM1 to target proteins through a dedicated E1–E2–E3 enzymatic cascade. Although ufmylation has been increasingly implicated in a range of human diseases, its role in endothelial cells (ECs) and vascular integrity remains largely unexplored. Here, we identify ufmylation as a critical regulator of vascular development and endothelial function. Bioinformatic analyses reveal that genetic variants within and adjacent to the Ufl1 locus, which encodes the UFM1-specific E3 ligase UFL1, are associated with multiple vascular pathologies. Constitutive, EC-specific deletion of Ufl1 in mice resulted in reduced vascular density and increased vascular permeability, leading to subepidermal hemorrhage, myocardial hypoplasia, growth retardation by embryonic day 16.5, and eventual perinatal lethality. Transcriptomic profiling of isolated ECs demonstrated that loss of UFL1 dysregulated gene programs essential for maintaining endothelial identity and barrier function—findings that were recapitulated in ufmylation-deficient human umbilical vein endothelial cells (HUVECs) following UFL1 or UFM1 silencing. Genetic inhibition of ufmylation in HUVECs suppressed proliferation, increased cell death, impaired tube formation, enhanced migration, and promoted endothelial-to-mesenchymal transition. Furthermore, tamoxifen-induced, EC-specific deletion of Ufl1 in adult mice led to cardiomyopathy as early as six weeks post-induction, characterized by increased left ventricular (LV) mass, LV dilation and reduced ejection fraction. Notably, endothelial ufmylation deficiency markedly exacerbated angiotensin II–induced pathological cardiac remodeling, accompanied by a significant reduction in coronary flow reserve, indicative of coronary microvascular dysfunction. Collectively, these findings establish endothelial ufmylation as an essential determinant of vascular development and homeostasis by promoting angiogenesis, preserving endothelial identity, and protecting against maladaptive cardiac remodeling