BACKGROUND: Endothelial dysfunction (ED) is involved in the development of atherosclerosis. Hesperidin, a citrus flavonoid with antioxidant and other biological properties, potentially exerts beneficial effects on endothelial function (EF). OBJECTIVE: We investigated the effect of hesperidin 2S supplementation on EF in overweight individuals. DESIGN: This was a randomized, double-blind, placebo-controlled study in which 68 individuals were randomly assigned to receive hesperidin 2S (450 mg/d) or a placebo for 6 wk. At baseline and after 6 wk of intervention, flow-mediated dilation (FMD), soluble vascular adhesion molecule-1 (sVCAM-1), soluble intracellular adhesion molecule-1 (sICAM-1), soluble P-selectin (sP-selectin), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were assessed. Acute, reversible ED was induced by intake of a high-fat meal (HFM). A second FMD scan was performed 2 h postprandially, and adhesion molecules were assessed 2 and 4 h postprandially. An additional exploratory analysis was performed in subjects with baseline FMD ≥3%. RESULTS: No significant change in fasting or postprandial FMD was observed after 6 wk of hesperidin intake compared with placebo intake. However, there was a trend for a reduction of sVCAM-1, sICAM-1, sP-selectin, SBP, and DBP after 6 wk of hesperidin treatment. In the FMD ≥3% group, hesperidin protected individuals from postprandial ED (P = 0.050) and significantly downregulated sVCAM-1 and sICAM-1 (all P ≤ 0.030). The results reported in the current article were not adjusted for multiplicity. CONCLUSIONS: Six weeks of consumption of hesperidin 2S did not improve basal or postprandial FMD in our total study population. There was a tendency toward a reduction of adhesion molecules and a decrease in SBP and DBP. Further exploratory analyses revealed that, in subjects with baseline FMD ≥3%, hesperidin 2S improved ED after an HFM and reduced adhesion molecules. These results indicate the cardiovascular health benefits of hesperidin 2S in overweight and obese individuals with a relatively healthy endothelium. This trial was registered at clinicaltrials.gov as NCT02228291.