Gestational diabesity is a metabolic condition where the mother shows pre-gestational obesity and develop gestational diabetes mellitus (GDM) (Cabalín et al., 2019). Human umbilical vein endothelial cells (HUVECs) from GDM pregnancies show Na+/H+ exchanger 1 (NHE1)-dependent alkaline intracellular pH (pHi). However, it is unknown whether gestational diabesity results in altered NHE1-dependent pHi modulation in HUVECs. Methods: HUVECs were isolated (collagenase digestion) from full-term pregnancies collected at the Clinical Hospital CHRISTUS-UC (Chile). The study conformed to the principles outlined in the Declaration of Helsinki. HUVECs were isolated from the following experimental groups: Normal pregnancies with maternal pre-pregnancy normal weight (Nnw, body mass index (BMI) 20-24.9 kg/m2, n = 5 or overweight (Now, BMI 25-29.9 kg/m2, n = 3), GDM pregnancies with maternal pre-pregnancy normal weight (GDMnw, n = 3), overweight (GDMow, n = 3) or obesity (GDMob or gestational diabesity, BMI >30 kg/m2, n = 3). Cells were cultured in medium 199 plus sera (20%) up to passage 2. The pHi was measured in cells loaded with the fluorescent pH-sensitive probe BCECF-AM (12 µM, 10 min) and exposed to NH4Cl (20 mM). Basal and pHi recovery rate (dpHi/dt) were estimated (up to 360 s) in cells exposed to 5 µM 5-N,N-hexamethylene-amiloride (HMA, NHE general inhibitor), 0.1 µM zoniporide (Zn, NHE1 inhibitor), 0.1 µM concanamycin A (CoA, V-ATPases inhibitor), or 10 µM Schering (Sch, H+/K+-ATPase inhibitor). Results: The basal pHi was higher (P<0.05, unpaired ANOVA) in HUVECs from GDMnw (7.73 ± 0.03, mean ± S.EM.), GDMow (7.53 ± 0.04), and GDMob (7.35 ± 0.03) compared with Nnw (7.08 ± 0.02) or Now (7.04 ± 0.02). The dpHi/dt was higher (P<0.05) in Now (2.82 ± 0.22 fold), GDMnw (4.01 ± 0.21), GDMow (4.33 ± 0.33), and GDMob (1.97 ± 0.06) compared with Nnw (0.0043 pHi units/10 s). The dpHi/dt in GDMob was lower than GDMnw and GDMow. Zn and HMA reduced the dpHi/dt in Nnw (18 ± 6 and 59 ± 9%, respectively), GDMnw (98 ± 2 and 95 ± 4%) GDMow (98 ± 2 and 94 ± 5%), and GDMob (58 ± 5 and 98 ± 2%); however, these inhibitors were not effective in cells from Now. Incubation of with CoA or Sch did not alter the dpHi/dt, but it was blocked in cells exposed to CoA or Sch in the presence of Zn, HMA, or both, in cells from GDMob. Conclusion: The pHi recovery in HUVECs from normal pregnancies is partially dependent on NHEs activity. However, in HUVECs from gestational diabesity the pHi recovery may require NHE1 along with other NHEs isoforms.