Background and aims: Myoinositol (Myo) favorably affects impaired glucose metabolism and insulin resistance, thus potentially improving vascular health. Endothelial Extracellular Vesicles (eEVs) release is considered an indicator of vascular damage. Aim of this study was to evaluate (both in vitro and in vivo) Myo effects on eEVs release. Materials and methods: In vitro studies: eEVs release after 16 hours exposure to Tumor Necrosis Factor-α (TNF-α, 1 ng/ml) was determined by flow cytometry in human umbilical vein endothelial cells obtained from women with gestational diabetes (GD-HUVECs) or from normo-glucose tolerant women (C-HUVECs) previously pre-incubated for 48 hours with Myo 0.1, 0.5 and 1 mM or alpha-lipoic acid (LA; 0.1-0.2 mM; positive control). In vivo studies: Circulating levels of EVs subtypes (from platelet, leukocyte, and endothelial cells) were evaluated by flow cytometry in freshly drawn whole blood from pregnant women with gestational diabetes (GDM) supplemented (GD+Myo, n=17) or not (GD, n=13) with Myo (up to 4 grams/day). Samples were collected at 29±2.6 weeks of gestation (gw), and before delivery (36±1 gw). Statistical analyses were performed using the Student’s t-test and analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test for post hoc comparisons. P values less than or equal to 0.05 were considered statistically significant. Results: In GD-HUVECs, pre-exposure to Myo significantly reduced TNF-α induced eEVs release (p<0.05). Moreover, in vivo supplementation with Myo (GD+Myo) was associated with a significant reduction in circulating eEVs (p<0.05) and with lower maternal weight gain during pregnancy (1.3±1.9 Kg vs 3.6±2.5 Kg in non-supplemented women, p=0.03) Conclusions: In GDM, Myo supplementation is associated with a reduction in markers of vascular damage (as indicated by the reduced eEVs concentrations) and with lower maternal weight gain. Myo supplementation might thus play a protective role against endothelial dysfunction, as furtherly suggested by the decreased TNF-α induced eEVs release observed in GD-HUVECs pre-exposed to Myo.