Background: There are conflicting results about the relationship between Vitamin D deficiency (VDD) and gestational diabetes mellitus (GDM). Aim: The aim of the study was to determine the association between 25 hydroxy vitamin D levels and GDM in Turkish Cypriot pregnant women. Study design: The authors conducted a prospective case-control study. Materials and Methods: The authors analysed serum 25OHD concentrations in 230 pregnant women between 2013-2014 in the winter period (November-April). Twentyfive hydroxy vitamin D was measured at the time of glucose tolerance testing at mid-gestation (24-26 weeks of pregnancy), in a population at increased risk for GDM. Patients were than divided in to two groups: pregnant women with GDM and pregnant women without GDM. Results: The mean level of 25 hydroxy vitamin D in the study group was 21.9 ±10.4 ng/ml. Pregnant women were diagnosed as having vitamin D insufficiency. The level of 25 hydroxy vitamin D in women with and without GDM were 22.3 ± 10.7 ng/dl and 20.3 ±9.3 ng/dl, respectively (p = 0.262). Conclusion: The present study showed 25OHD insufficiency in pregnant women at midgestation which was not associated with GDM.