Gestational diabetes mellitus (GDM), is the most common medical complications of pregnancy. This study aimed to clarify the effect of second-trimester vitamin D deficiency on the 75 g oral glucose tolerance test (OGTT) screening and insulin resistance. A total of 120 pregnant women with a singleton pregnancy at a gestational age of 26-28 weeks were analyzed. Participants were divided into two groups according to 25-hydroxyvitamin D levels; vitamin D deficiency, and control groups. For GDM scan, 75 g OGTT was preferred. GDM prevalence was 17.5% in vitamin D deficiency group and 13.75% in control group, there is no significant difference in GDM prevalence (p = 0.149). Fasting plasma glucose and 1-h plasma glucose levels were significantly higher in the vitamin D deficiency group than in the control group (p < .001 and p < .001, respectively). No significant differences were observed between 2-hour plasma glucose levels (p = .266). The HOMA-IR level was significantly higher in the vitamin D deficiency group than in the control group (p < .001). The findings of the present study suggested that vitamin D deficiency in the second trimester was inversely correlated with fasting and 1-h plasma glucose after 75 g glucose challenge test; also, low 25 OHD3 levels were associated with insulin resistance.