Reduced adenosine uptake via human equilibrative nucleoside transporters 1 (hENT1) in human umbilical vein endothelial cells (HUVECs) from gestational diabetes (GD) is reversed by insulin by restoring hENT1 expression (Westermeier et al. 2015). In addition, GD-increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1) is modulated by insulin in this cell type (Guzmán-Gutiérrez et al. 2012). Insulin modulation of L-arginine transport idepends on expression and activation of A2A adenosine receptors (A2AAR) (Guzmán-Gutiérrez et al. 2012). We studied whether the reversal of the associated effects of GD on transport by insulin depends on adenosine receptors expression and activation. hENT1 and hCAT-1 expression (mRNA number of copies, protein abundance) and activity (transport kinetics), and A1AR, A2AAR and IR-A/IR-B expression in primary cultures of HUVECs from normal (n = 68) or GD (n = 68) pregnancies were assayed. Overall adenosine (0.15-500 μM adenosine) and L-arginine (0.75-1000 μM L-arginine) transport was measured 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 A1AR, A2AAR, IR-A, IR-B and IR-A/B knockdown cells in the absence or presence of insulin (0-10 nM, 8 h), CPA (A1AR agonist, 30 nM), DPCPX (A1AR antagonist, 30 nM), CGS-21680 (A2AAR agonist, 30 nM), ZM-241385 (A2AAR antagonist, 10 nM). Values are mean ± SEM (n = 27-31 different cell cultures). Results show that GD associates with higher mRNA and protein abundance of hCAT-1 (1.9 ± 0.3 and 2.2 ± 0.2 fold, respectively) (P<0.03, ANOVA), but reduced mRNA and protein abundance of hENT1 (67 ± 12 and 73 ± 15%, respectively) (P<0.05) compared with normal pregnancies. GD also increased mRNA and protein abundance for A1AR (3.7 ± 1.1 and 4.5 ± 0.8 fold, respectively) and IR-A mRNA (1.7 ± 0.4 fold) (P<0.04). The Vmax for hENT1 adenosine transport was reduced (P<0.02) in GD (2.71 ± 0.12 pmol/μg protein/s) compared with normal (1.01 ± 0.03 pmol/μg protein/s) pregnancies. The Vmax for hCAT-1 L-arginine transport was increased (P<0.03) in GD (3.91 ± 0.41 pmol/μg protein/min) compared with normal (1.29 ± 0.28 pmol/μg protein/min) pregnancies. However, apparent Km values were not significantly (P>0.05) altered. Insulin reversed the GD-associated changes in hENT1 and hCAT-1 expression and activity, and changes in A1AR, A2AAR, and IR-A/IR-B mRNA expression to values in normal pregnancies. However, insulin effects were abolished in A1AR, IR-A or IR-A/B knockdown cells. In conclusion, insulin requires normal A1AR and IR-A expression to restore GD-reduced hENT1 and hCAT-1 expression and activity in HUVECs.