Prenatal repair of open spina bifida (OSB) via the percutaneous fetoscopic approach does not require maternal laparotomy, hysterotomy, or exteriorization of the uterus. This technique does require intrauterine carbon dioxide (CO2) insufflation for surgical manipulation of the fetus. There is limited data on the physiological effects of CO2 insufflation on human fetuses and no data using the entirely percutaneous fetoscopic surgical technique. Our aim was to examine the effects of CO2 insufflation on fetal blood gases after percutaneous fetoscopic OSB repair. Prospective study of patients that underwent percutaneous fetoscopic OSB repair from 2/2019 to 7/2020. Fetal cordocentesis of the umbilical vein was performed in cases with favorable access to the umbilical cord. The umbilical vein cord blood samples were obtained under ultrasound guidance immediately at the conclusion of the OSB repair. Simultaneous maternal arterial blood gas samples were also obtained. Results are reported as median (range). Of 20 patients who underwent percutaneous fetoscopic OSB repair, 7 patients (35%) underwent fetal blood sampling. Gestational age at time of surgery was 27.4 (24.0 – 27.9) weeks and operative time was 183 (156 – 251) minutes. The CO2 exposure time was 122 (57 – 146) minutes with maximum pressure of 13.5 (12.0 – 15.0) mmHg. Fetal umbilical vein results were: pH 7.346 (7.301 – 7.387), pO2 56.2 (47.1 – 99.9) mmHg, pCO2 43.8 (36.2 – 53.0) mmHg, HCO3 23.9 (20.1 – 25.6) mmol/l and base excess (BE) -2.2 (-4.5 – -0.4) mmol/l. The lowest fetal pH recorded was 7.301. Simultaneous maternal arterial blood gas results were: pH 7.370 (7.284 – 7.420), pO2 187.5 (124.4 – 405.2) mmHg, pCO2 36.6 (30.7 – 46.0) mmHg, HCO3 21.3 (18.0 – 22.8) mmol/l and BE -3.2 (-5.9 – -1.8) mmol/l. After prolonged CO2 insufflation of the uterus, the fetal umbilical vein pH remained within normal range, above levels associated with fetal acidosis.