Aim: A 50 g glucose challenge test (GCT) is recommended for screening all pregnant women for gestational diabetes mellitus. In this study, the effect of GCT on the thiol/disulfide balance was investigated. Methods: One-hundred women that underwent a 50 g GCT at 24–28 weeks of gestation (63 positive and 37 negative results) were evaluated in terms of thiol/disulfide in serum samples at test hours 0 and 1. Results: Compared to the baseline values (hour 0), after the glucose load (hour 1), the thiol and native thiol/total thiol (p < 0.0001) of the GCT-positive women were reduced whereas the values of glucose, disulfide, disulfide/native thiol, disulfide/total thiol (p < 0.0001) and total thiol increased (p = 0.018). Conclusion: In GCT-positive pregnant individuals, the glucose load increases oxidative stress by changing the thiol/disulfide homeostasis. Such an effect is not observed in healthy pregnancies.