Objective: To investigate the role and relationship of serum Vitamin B12 and Glutathione peroxidase in pregnancy with gestational diabetes mellitus (GDM).
 Study Design: Comparative cross-sectional study.
 Place and Duration of Study: Physiology Department, University of Health Sciences, Lahore Pakistan, from Feb 2019 to Mar 2020.
 Methodology: Ninty pregnant women were recruited, fulfilling the inclusion and exclusion criteria. Women with GDM were the cases, and those without GDM were the controls. Serum Vitamin B12 and Glutathione peroxidase were measured by ELISA.
 Results: Serum Vitamin B12 was significantly lower in cases compared to controls (158.98±7.38 and 357.48±42.07mg/dL, respectively, at p<0.001. Glutathione peroxidase was also significantly lower in cases compared to controls (2.68±1.13U/mL and 5.53±1.05U/mL, respectively, at p<0.001. Correlation analysis showed that Vitamin B12 and Glutathione peroxidase had a significant positive correlation in cases (p<0.001 and rho=0.76) and a non-significant correlation in controls (p=0.87 and rho=-0.024). Linear regression analysis showed that Vitamin B12 positively affects Glutathione peroxidase (β=0.13, p<0.001).Vitamin B12 and Glutathione peroxidase have a significant, negative effect on fasting blood glucose (β =-0.06, p<0.001 and β=-3.66, p<0.001, respectively).
 Conclusion: Deficient Vitamin B12 leads to reduced Glutathione levels in GDM pregnancy. A deficiency of these two predicts elevated blood sugar levels.