Background: Gestational diabetes mellitus (GDM) is associated with both maternal and perinatal morbidity and mortality. Studies have suggested that Vitamin D plays a major role in pancreatic beta-cell function and insulin sensitivity and this has been associated with type 2 Diabetes Mellitus (T2DM). Despite conflicting reports on the association between GDM and Vitamin D insufficiency, Vitamin D supplementation in pregnancy is being practised in some developed countries to avert possible sequelae of inadequacy of Vitamin D. This study was aimed at investigating the relationship between Vitamin D insufficiency and GDM in this environment to provide evidence of an association or otherwise. Methods: This was a case-control study. Two hundred (200) pregnant women that were referred for Oral Glucose Tolerance Test (OGTT) were recruited from the Department of Chemical Pathology of the National Hospital Abuja. They were made up of 100 cases and 100 controls. Cases were defined as pregnant women with a fasting, 1 hour or 2 hour plasma glucose value of ≥ 5.1 mmol/L, ≥ 10.0 mmol/L or ≥ 8.5 mmol/L respectively (i.e. diagnosed with GDM) that met the eligibility criteria. Controls were those whose corresponding plasma glucose levels fell below the above stated values. Socio demographic and clinical data were obtained using a semi-structured interviewer administered questionnaire. Plasma total calcium, phosphate, albumin, 25 hydroxycholecalciferol and intact parathyroid hormone (PTH) were determined. The main statistical measures were the odds ratio for GDM among women with Vitamin D insufficiency and the adjusted odds ratio after logistic regression using variables that serve as possible effect modifiers or confounders in the association between Vitamin D insufficiency and GDM.