Plant-derived extracellular vesicles (PDEVs) are small lipoproteic structures released from plant cells which recently have garnered growing attention for their biological properties. In this context, several in vitro and in vivo studies have demonstrated the anti-inflammatory and anti-oxidant role of PDEVs isolated from the Citrus limon L. juice (L-EVs). Nevertheless, the effects of L-EVs on endothelial functions have never been explored. Hence, this study aimed to investigate the potential anti-inflammatory role of L-EVs in endothelial cells isolated from the umbilical cord vein of women with gestational diabetes (GD-HUVECs) as a suitable in vitro model able to reproduce the pro-inflammatory phenotype of a diabetic endothelium. To this aim, GD-HUVECs and relative controls derived from healthy women (C-HUVECs) were pre-treated for 24 h with L-EVs (10 and 25 µg/mL) and then stimulated for 16 hours with the pro-inflammatory stimulus TNF-α (10 ng/mL). Firstly, as observed via confocal microscopy analyses, results showed the ability of C-and GD-HUVECs to internalize L-EVs following 4 hours of incubation. Subsequently, we found that in both C- and GD-HUVECs, the pre-treatment with L-EVs reduced vascular adhesion molecule-1 (VCAM-1) protein expression and membrane exposure evaluated through flow cytometry and IncuCyte® imaging system, respectively. Reduced VCAM-1 exposure was also associated with a significant reduction of monocytes-HUVECs adhesion. Taken together, these data demonstrated that L-EVs exert an anti-inflammatory role in our cellular model and paves the way for further studies investigating their potential use in the prevention of vascular endothelial dysfunction associated with diabetes.