Abstract Body: The gap junction protein connexin 43 (Cx43) has been associated with human pathological vascular smooth muscle cell (SMC) proliferation and neointima formation. We previously identified that mitogen-activated protein kinase (MAPK) phosphorylation of Cx43 results in binding with the cell cycle protein cyclin E, facilitating neointima formation in mice. However, the specific nature of these interactions and their relevance to human disease has never been described. We aimed to define protein interaction sites for Cx43 and cyclin E and develop novel compounds to disrupt their effects on SMC. Using an ex vivo human saphenous vein model of neointima formation, we identified increased MAPK-phosphorylated Cx43 and cyclin E-Cx43 interactions in human explant tissues. We used peptide arrays to define a cyclin E-Cx43 binding region and generated ‘CycliCx’, a stearate-linked phospho-mimetic peptide. CycliCx inhibits the effects of PDGF-β treatment on human SMC, altering Cx43 trafficking and interactions with cyclin E and reducing SMC proliferation and migration. RNAseq analysis identified that CycliCx significantly inhibits PDGF-β-induced proliferative pathways in SMC and limits proliferation. CycliCx limits neointima formation in mice in vivo and in ex vivo human saphenous vein explants. Our data provide the strongest evidence to date, showing mechanistic regulation of SMC proliferation by the Cx43 protein and identifying a significant potential therapeutic strategy for preventing neointimal formation.