Gestational diabetes mellitus (GDM) associates with endothelial dysfunction of fetoplacental endothelium. Human umbilical vein endothelial cells (HUVECs) from GDM pregnancies show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1) and nitric oxide (NO) synthesis by the endothelial NO synthase (eNOS). Exosomes modulate different cellular functions; however, it is unknown whether endothelial-derived exosomes modulate L-arginine transport and NO synthesis (i.e., the L-arginine/NO signalling pathway) in HUVECs. Exosomes were isolated from primary cultures of HUVECs from normal or GDM pregnancies after 48 hours of incubation with exosomes-free media (Théry et al. Curr Protoc Cell Biol, 2006). Exosomes were characterized by density floating and protein markers (Western blotting). HUVECs were incubated (8 or 24 hours) with 1, 5 or 10 μg exosomes derived from HUVECs from normal (n = 8) or GDM (n = 8) pregnancies. hCAT-1 expression (protein abundance) and L-arginine uptake (150 mmol/L, 3 μCi/mL L-[3H]arginine, 37°C, 1 minute), and total (eNOS) and Ser1177 phosphorylated eNOS (P~Ser1177eNOS) were assayed. Values are mean ± SEM (n = 8 different cell cultures), compared by ANOVA. GDM associated with increased of L-arginine transport (2.2 ± 0.3 vs 1.1 ± 0.2 pmol/mg protein/minute), respectively. hCAT-1 (1.8 ± 0.2 fold) protein abundance and P~Ser1177eNOS/eNOS (1.9 ± 0.3 fold ) compare with cells from normal pregnancies. The results also show that 10 mg exosomes from cells from normal pregnancies reversed (P<0.05) GDM-effect on L-arginine transport, hCAT-1 protein abundance and P~Ser1177eNOS/eNOS. However, exosomes from GDM pregnancies did not alter (P>0.05) L-arginine transport, hCAT-1 expression and P~Ser1177eNOS/eNOS in HUVECs from normal pregnancies. Additionally, these parameters were unaltered when HUVECs from normal or GDM pregnancies were incubated with exosomes from cells from GDM pregnancies. In conclusion, the results show that exosomes from HUVECs from normal pregnancies could have a protective effect in GDM-associated alterations on endothelial L-arginine/NO signalling pathway.