Endothelial cell (EC) dysfunction is classically associated with subclinical atherosclerosis and the development of atheroprone lesions. Extracellular vesicles (EVs) are biological nanoparticles secreted by most cell types that contain a complex mix of cell-type specific signalling proteins and nucleic acids. At high concentrations, alcohol is a known risk factor for atherosclerosis but may be protective at lower concentrations. In this study, we first investigated whether alcohol (EtOH) at varying concentrations can alter the number and/or phenotype of EVs released from endothelial cells before the proteomic profile of endothelial cell-derived EVs was assessed. Human aortic endothelial (HAEC) and smooth aortic vascular muscle cells (HuSMCs) were first characterised for lineage specific markers before endothelial cell dysfunction was induced following treatment of HAECs with low and high doses of EtOH. EVs released from HAEC were isolated from conditioned cell media, enumerated and characterised using dynamic light scattering (DLS), immunoblot and AmnisTM Cell Stream FACS analysis for EV biomarkers before the proteomic profile of the EVs was evaluated using Liquid chromatography massspectrometry (LC-MS) analysis. The data generated suggest that the number of endothelial derived-EVs released following endothelial dysfunction increases following exposure of cells to different concentrations of EtOH. Moreover, the protein cargo within these EVs varies, even though the EVs originated from the same cell source. Proteomic and bioinformatic analysis revealed that protein metabolism and cell growth and/or maintenance are the key pathways and biological processes associated with these EVs and provides further insight into elucidating their putative role following endothelial dysfunction. Lastly, EVs from dysfunctional HAECs had little effect on the growth and migration of HuSMCs but did promote HuSMC de-differentiation following treatment with 25-EXO. These data highlight the potential important role for EVs released following endothelial dysfunction on the underlying smooth muscle cell population that may contribute to arteriosclerotic lesion formation