Obesity during pregnancy (OP) is a condition where mothers with normal body mass index (BMI) develop supraphysiological weight gain during pregnancy ending with obesity (BMI ≥ 30) (1). Increased risk of gestational diabetes (GD) is associated with OP (~2.6 fold). GD is a condition associated with metabolic alterations of the placental endothelium, leading to altered vascular reactivity (2). Even when obesity is correlated with high cardiovascular risk, and GD associates with reduced equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine transport (3), nothing is known regarding the potential effect of OP on the human fetoplacental endothelial function. We evaluated whether OP associates with altered nitric oxide (NO) synthesis and whether this pathological condition worsens the GD-reduced hENT1-adenosine transport in human umbilical vein endothelial cells (HUVEC). Umbilical vein reactivity in the absence/presence of 10 µM L-NAME in response to increasing insulin concentrations (10-10-10-6 M) were measured in umbilical vein rings from normal and OP groups in a wire myograph. L-[3H]Citrulline formation (100 µM L-arginine, 2.7 µCi/ml, 37C, 30 min), adenosine transport (31-250 µM Adenosine, 3 µCi/ml, 25C, 20 sec) in the absence/presence of 1 or 10 µM nitrobenzylthioinosine, hENT1 and endothelial NO synthase (eNOS) protein and mRNA expression and eNOS phosphorylation, were measured in primary cultures of HUVEC from normal (N) or GD pregnancies coursing without (N or GD groups) or with (OP or GD+OP groups) OP. Insulin-mediated vasodilation was almost absent in vein rings from OP. L-Citrulline formation was only decreased in OP (~96%) in HUVEC. Moreover, eNOS expression is reduced in OP (~42 and 80% for protein abundance and mRNA expression, respectively), but only protein abundance is lower in GD (~38%) compared to N. The mRNA expression was also reduced (~90%) in GD+OP. Higher inhibitory (p-Thr495) phosphorylation of eNOS was found in OP compared with the other groups. Adenosine transport via hENT1 is reduced in OP, GD and GD+OP compared with N group (Vmax/Km: 0.25±0.04* N; 0.11±0.01 OP; 0.15±0.03 GD; 0.005±0.02 GD+OP, 1way ANOVA;*p<0.05). However hENT1 protein abundance and mRNA expression are increased in OP (~1.7 and ~3-fold, respectively), but reduced in GD (~60%) and GD+OP (~50%) compared with N group. In conclusion, OP is a pathological condition that results in lower NO synthesis; meanwhile in women with OP and GD inhibition of adenosine transport is likely due to reduced hENT1 transport activity via mechanisms that is independent of NO synthesis.