Abstract Body: Endothelial cell (EC) dysfunction is a primary cause of many cardiovascular disorders. While numerous epigenetic and transcriptional regulators of endothelial homeostasis have been identified, the post-translational mechanisms that preserve EC function and vascular integrity remain poorly understood. Neddylation is a highly conserved post-translational modification that covalently conjugates the ubiquitin-like protein NEDD8 to target substrates through a NEDD8-specific E1–E2–E3 enzymatic cascade. Here, we define the functional importance of neddylation in endothelial integrity and vascular development and identify its key downstream effectors. Constitutive EC-specific deletion of Nae1, encoding NAE1—the regulatory subunit of the sole NEDD8 E1 enzyme (initiate neddylation process), using Cdh5-Cre resulted in early embryonic lethality by E11.5 due to defective vasculogenesis. In contrast, tamoxifen-induced deletion of Nae1 in adult mice via Cdh5-^CreERT2 led to loss of EC identity, enhanced EC transdifferentiation, and EC death, resulting in increased vascular permeability across multiple vascular beds, hemorrhage, multi-organ failure (including lung and liver), and premature lethality within ~8 weeks post induction. Cullin family proteins (CUL1-9) are predominantly neddylated in ECs and upon neddylation, serve as scaffolds for the assembly of multi-subunit Cullin-RING ubiquitin ligases (CRLs). Among these, CUL3 is the most highly expressed cullin in ECs. Although mutations in CUL3 and its binding partner LZTR1 are linked to hypertension and vascular leakage, their roles in maintaining vascular function and integrity remain largely unexplored. We found that constitutive EC–specific deletion of Cul3 causes early embryonic lethality by E11.5 with defective vasculogenesis, recapitulating the phenotype observed in NAE1-deficient embryos. In vitro data in HUVECs revealed that depletion of CUL3 impaired wound healing, tube formation and proliferation of ECs but promoted trans well migration. Transcriptomic analysis of HUVECs revealed that loss of CUL3 suppressed endothelial signature gene expression and promoted EC transdifferentiation, findings validated by qPCR, immunoblotting, and complementary biochemical assays. Collectively, our data identify neddylation and its downstream effector CUL3 as critical regulators of vascular development and endothelial function by maintaining EC identity and restraining pathological EC transdifferentiation and cell death.