Background: This meta-analysis aimed to pool all the available data to provide a well-powered assessment of the role of maternal Vitamin D levels in developing gestational diabetes mellitus (GDM) because already published studies evaluating this association are small in sample size and yielded conflicting findings. Material and Methods: A systematic review and meta-analysis of observational studies was performed. We searched electronic databases (PubMed and Cochrane Central) from inception to April 2021 for published and unpublished observational studies that determined the association between the reduction of Vitamin D levels and the risk of developing GDM in pregnant women. Results from studies were pooled as mean ± standard deviation (SD) and odds ratios (OR) using the random-effects model. Results: Forty-four studies, consisting of 37,838 pregnant women were included in this meta-analysis. Dichotomous studies showed a significant association between maternal Vitamin D deficiency and increased risk of GDM (OR = 1.38; 95% confidence interval [CI] = 1.21-1.57; P < 0.00001). Studies with continuous data also showed a significant association between maternal Vitamin D deficiency and the risk of developing GDM (weighted mean difference (WMD): –5.14 nmol/L, 95% CI = –6.28 to -4.00; P < 0.00001). Moderate heterogeneity was also detected. Conclusion: In conclusion, all studies demonstrated that lower levels of maternal serum Vitamin D were associated with a higher risk of developing GDM in pregnancy.