Gestational diabetes mellitus (GDM) associates with maternal hyperglycaemia, foetoplacental endothelial dysfunction, and impaired insulin signaling (1). Women with GDM treated with diet (GDMd) show normal glycaemia at delivery (2). However, some patients do not reach the recommended glycaemia with diet and they are passed to insulin therapy (GDMi). Human umbilical vein endothelial cells (HUVECs) from GDMi show increased L-arginine transport via the human cationic amino acid transporters 1 (hCAT-1), endothelial nitric oxide synthase (eNOS), and insulin receptor A (IR-A) (3). GDMi alterations were reversed by treating these cells in vitro with insulin (i.e., exogenous insulin). However, the cell signaling triggered by exogenous insulin in HUVECs from GDMi is unknown. We studied whether insulin signaling via the 44 and 42 kDa mitogen-activated protein kinases (p44/42mapk) and protein kinase B/Akt (Akt) is altered in HUVECs from GDMi. HUVECs were isolated from full term normal (n = 13), GDMd (n = 14), or GDMi (n = 18) pregnancies collected at the Clinical Hospital UC-CHRISTUS and Hospital San Juan de Dios (HSJD) (Chile) after informed consent approved by the UC-CHRISTUS and HSJD ethics committees. The investigation conforms to the principles outlined in the Declaration of Helsinki. IR-A, insulin receptor B (IR-B), hCAT-1, and eNOS mRNA expression were evaluated by qPCR. Phosphorylation of p44/42mapk and Akt were evaluated by Western blot. L-Arginine transport kinetics was measured in Krebs solution and the NO level was evaluated in 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM)-loaded cells in the absence or presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 µmol/L, NOS inhibitor). Experiments were performed in the absence or presence of exogenous insulin (1 nmol/L, 8 h), Akt inhibitor IV (1 µmol/L, Akt inhibitor, Inh IV), and PD-98059 (10 µmol/L, mitogen-activated protein kinase kinase 1/2 inhibitor). Values are means ± S.E.M., compared by ANOVA, P<0.04. IR-A but not IR-B mRNA expression was higher (2.6 ± 0.5 fold) and phosphorylated p44/42mapk protein abundance was higher (1.7 ± 0.1 fold) but phosphorylated Akt was unaltered in GDMi compared with normal pregnancies but similar to cells from GDMd pregnancies. In the absence of exogenous insulin, PD-98059 but not Inh IV partially blocked (33 ± 0.32%) the GDMi-increased maximal L-arginine transport capacity (Vmax/Km) but none of these inhibitors altered the GDMi- and GDMd-increased NO level, eNOS, and hCAT-1 mRNA expression. Exogenous insulin blocked the GDMi and GDMd effects on NO level, eNOS, and hCAT-1 mRNA expression, an effect inhibited by the Inh IV but not PD-98059. In conclusion, insulin beneficial effect on HUVECs from GDMi requires Akt activity.