Abstract Body: Cardiovascular disease is the major cause of mortality in people with type 2 diabetes, primarily due to endothelial dysfunction. Recent studies have suggested that transcriptional reprogramming plays a central role in endothelial dysfunction, but its regulation in the context of diabetes remains poorly understood. In this study, we utilized 3D chromatin mapping analysis (H3K27ac HiChIP) to investigate the transcriptional regulation in aortic endothelial cells (db/db mice) and HUVEC cells treated with high glucose (HUVEC^high-glu). We found that these cells were characterized by a massive loss of enhancer-promoter loops, which was associated with transcriptional downregulation. Through transcription factor motif scan, ChIP-seq and ATAC-seq analysis, we uncovered that the lost loop anchors are demarcated by reduced JUNB (transcription factor) binding while their chromatin accessibility remains unchanged. Moreover, we demonstrated that overexpression of JUNB significantly restored enhancer-promoter loops lost in HUVEC^high-glu and ameliorated endothelial dysfunctions both in vitro (cell scratch, Transwell), ex vivo (aorta relaxation/contraction) and in vivo (hindlimb ischemia) assays. We further showed that JUNB protein (but not RNA) expression is reduced in aortic endothelial cells (db/db), HUVEC^high-glu and primary endothelial cells from diabetic patients. Through RIME (Rapid immunoprecipitation mass spectrometry of endogenous protein) assay, we identified that RBBP6 (RB Binding Protein 6, Ubiquitin Ligase) is a JUNB interacting protein in HUVEC^high-glu. We further showed that overexpression of RBBP6 downregulated JUNB expression while proteasome inhibitor MG132 treatment largely abolished the inhibitory effect of RBBP6 on JUNB protein levels in normal HUVEC cells, suggesting that RPPB6 promoted JUNB degradation through ubiquitination. Notably, knockdown of RBBP6 in HUVEC^high-glu significantly upregulated JUNB expression, enhancer-promoter loopings and improved endothelial dysfunction. Overall, our findings provide novel 3D chromatin insights into diabetic endothelial dysfunction and illustrate a model whereby RBBP6-mediated JUNB degradation leads to global loss of enhancer-promoter looping and transcriptional inhibition in diabetic endothelial dysfunction.