Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. In our previous study, a panel of miRNA including miR-134-5p was deregulated in young acute coronary syndrome (ACS) patients. However, the roles of these ACS-associated miRNAs in endothelial dysfunction, an early event preceding atherosclerosis, remain to be investigated. In the present study, human aortic endothelial cells (HAECs) were treated with 7-ketocholesterol (7-KC) to induce endothelial dysfunction. Following treatment with 20 μg/ml 7-KC, miR-134-5p was significantly up-regulated and endothelial nitric oxide synthase (eNOS) expression was suppressed. Endothelial barrier disruption was evidenced by the deregulation of adhesion molecules including the activation of focal adhesion kinase (FAK), down-regulation of VE-cadherin, up-regulation of adhesion molecules (E-selectin and ICAM-1), increased expression of inflammatory genes (, and ) and AKT activation. Knockdown of miR-134-5p in 7-KC-treated HAECs attenuated the suppression of eNOS, the activation of AKT, the down-regulation of VE-cadherin and the up-regulation of E-selectin. In addition, the interaction between miR-134-5p and mRNA was confirmed by the enrichment of transcripts in the pull-down miRNA-mRNA complex. Knockdown of miR-134-5p increased expression whereas transfection with mimic miR-134-5p decreased FOXM1 protein expression. In summary, the involvement of an ACS-associated miRNA, miR-134-5p in endothelial dysfunction was demonstrated. Findings from this study could pave future investigations into utilizing miRNAs as a supplementary tool in ACS diagnosis or as targets for the development of therapeutics.