Background: Gestational diabetes mellitus (GDM) is described as varying degrees of glucose intolerance that occur within pregnancy. The worldwide prevalence of GDM accounts for 4 to 28 percent, depending on the ethnic composition of the population and the diagnostic procedures used. The aim of the study was to assess the serum concentration of vitamin D in pregnant women with GDM. Methods: This cross-sectional study involved a group of pregnant women with GDM cases and a group of apparently non-diabetic pregnant women (control group). The duration of the study ranged from 6-12 months. Serum vitamin D levels were measured in both groups. Result: Pregnant women with GDM had significantly decreased serum vitamin D than the control group. Among pregnant women with GDM, 40% had deficient serum vitamin D (<10 ng/ml), and 55.6% had insufficient value (10–30 ng/ml). The deficient serum vitamin D was significantly higher in pregnant women with GDM, but insufficient vitamin D was statistically nonsignificant, while sufficient serum vitamin D was significantly higher in healthy pregnant women. There was no statistically significant difference between pregnant women with GDM and the control group regarding the demographic data and lifestyle except for the statistically significant association between lower income and decreased serum concentration of vitamin D. Conclusion: Pregnant women with GDM have reduced serum concentration of vitamin D. This finding might help with GDM prediction.