Background/Aims: The aim of this prospective study was to determine ionized and total magnesium (Mg) levels in pregnant subjects with and without gestational diabetes mellitus (GDM). Methods: Eighty-five women, 26-28 weeks pregnant, were recruited for routine oral glucose tolerance tests (OGTT); 45 had normal OGTT results and 40 were diagnosed with GDM. Electrolyte levels, including ionized and total Mg, were analyzed. Results: Gestational age and BMI were similar between the two groups (p = 0.800, p = 0.025). Multivitamin use was higher in the control group (p = 0.036). Fasting blood glucose was higher in the GDM group (p < 0.001). The median total Mg levels were 1.9 mg/dl (range 1.6-2.2) in the control group and 1.8 mg/dl (range 1.2-2.1) in the GDM group (p < 0.001). The median ionized Mg levels were 0.5 mmol/l (range 0.4-0.6) in the control group and 0.4 mmol/l (range 0.4-0.5) in the GDM group (p < 0.001). Conclusion: Our study revealed a relationship between low total and ionized Mg levels and GDM, as in type 2 diabetes mellitus (DM). The literature regarding type 2 DM and our findings suggest that Mg is the key ion in the pathophysiology of GDM. Low-dose Mg supplementation was not related to GDM; however, pharmacological doses in the various stages of pregnancy could be beneficial and should be investigated.