Gestational diabetes (GD) associates with human placenta endothelial dysfunction (Guzmán-Gutiérrez et al. 2014). We reported that human umbilical vein endothelial cells (HUVEC) from GDM exhibit reduced adenosine uptake via human equilibrative nucleoside transporters 1 (hENT1), and that insulin restores hENT1 expression to values in HUVEC from normal pregnancies (Westermeier et al. 2011). Insulin receptor isoforms A (IR-A) and B (IR-B) are expressed in HUVEC, and GDM increases IR-A mRNA expression compared with normal pregnancies (Westermeier et al. 2011). We assayed whether insulin reversal of GDM effects depend on restoring IR-A expression and its associated cell signalling. hENT1 expression (mRNA number of copies, protein abundance) and activity (transport kinetics), and the role of IR-A/IR-B expression and signalling (total (p42/44mapk) and phosphorylated p42/44mapk (P~p42/44mapk) and total (Akt) and phosphorylated Akt (P~Akt)) in primary cultures of HUVEC isolated from normal (n = 33) or GDM (n = 33) pregnancies were assayed. Overall adenosine transport was measured (0.15-500 µM adenosine) in the absence or presence of 1 µM S-(4-nitrobenzyl)-6-thio-inosine (NBTI, ENT1 inhibitor), 2 mM hypoxanthine (ENT2 substrate), or both. Experiments were in IR-A, IR-B and IR-A/B knockdown cells. Cells were incubated in the absence or presence of insulin (0-10 nM, 8 h), PD-98059 (10 µM, MAPK kinase inhibitor) or wortmannin (30 nM, PI3K inhibitor). Values are mean ± SEM (n = 22-25 different cell cultures). Results show that GDM associates with higher (1.6 ± 0.2 fold) IR-A mRNA (P<0.04, ANOVA) with not changes (P>0.05) in IR-B mRNA expression. In addition, P~p42/44mapk/p42/44mapk and P~Akt/Akt ratios were lower (47 ± 6 and 29 ± 4%, respectively) in GDM compared with normal pregnancies. The Vmax for hENT1-adenosine transport was reduced (P<0.02) in GDM (2.82 ± 0.17 pmol/μg protein/s) compared with normal (1.12 ± 0.04 pmol/μg protein/s) pregnancies. However, apparent Km values were not significantly (P>0.05) altered (43 ± 9 or 36 ± 4 µM for normal or GDM, respectively). Insulin reversed the GDM-reduced hENT1 expression and activity, and GDM-increased IR-A/IR-B mRNA expression and P~p42/44mapk/p42/44mapk and P~Akt/Akt ratios to values in cells from normal pregnancies. However, insulin effects were abolished in IR-A or IR-A/B knockdown cells. In conclusion, insulin requires normal IR-A expression and p42/44mapk and Akt signalling to restore GDM-reduced hENT1 expression and activity in HUVEC from GDM.