<p>Methylphenidate (MPH) is a widely used psychostimulant for treating attention-deficit/hyperactivity disorder (ADHD). Emerging epidemiological findings suggest that MPH treatment increases the risk of cardiovascular events. However, as causality could not be inferred, further investigation of the potential impact of MPH on human vascular endothelial function is warranted. We investigated the effects of MPH on human brain microvascular endothelial cells (HBEC) and human aortic endothelial cells (HAEC) across a range of concentrations, including those corresponding to therapeutic plasma levels, at 24 and 48 h. Gene expression profiling of twenty-nine endothelial function-related genes was performed. Protein expression was assessed by ELISA, Western blotting, immunofluorescence and FACS, and barrier function was evaluated using FITC-dextran permeability and electric cell-substrate impedance sensing assays. Key endothelial markers with differential levels were analysed also in plasma from ADHD children (<i>n</i> = 87) and adults (<i>n</i> = 102), and matched controls (<i>n</i> = 4 and <i>n</i> = 44, respectively). Exposure to MPH altered levels of several endothelial function-related genes. Across all concentrations tested, MPH increased the secretion of von Willebrand factor (vWF) and tissue plasminogen activator, whereas supratherapeutic concentrations 50 µM and/or 100 µM MPH decreased Claudin-5 expression and impaired endothelial barrier integrity. In clinical plasma samples, children receiving MPH showed higher vWF levels compared with medication-naïve children, but this was not observed in adult samples. Our findings suggest that acute exposure to therapeutic MPH concentration was associated with endothelial activation, and that supratherapeutic concentrations (50 µM and/or 100 µM) may further compromise barrier integrity. These experimental outcomes may propose potential effects on vascular health associated with MPH use. Further studies are needed to confirm these findings.</p>