BACKGROUND Gestational diabetes mellitus (GDM) is a common complication of pregnancy, with significant short-term and long-term implications for both mothers and their offspring. Previous studies have indicated the potential benefits of vitamin D in reducing the risk of GDM, yet little is known about this association in twin pregnancies. This study aimed to investigate maternal vitamin D status in the second trimester and examine its association with the risk of GDM in twin pregnancies. METHODS We conducted a prospective cohort study based on data from the Chongqing Longitudinal Twin Study (LoTiS). Peripheral blood serum was collected from the mothers in the second trimester to measure 25(OH)D concentrations. GDM was diagnosed at 23-26 weeks of gestation using a 75-g 2-h oral glucose tolerance test. We used multivariable logistic regression analyses to examine the correlations between vitamin D status and the risk of GDM. RESULTS Of the total participants, 93 (29.9%) women were diagnosed with GDM. The mean serum 25(OH)D concentration in the second trimester was 31.1 ± 11.2 ng/mL, and the rate of vitamin D insufficiency and deficiency were 23.5% and 18.7%, respectively. Compared to women with a 25(OH)D concentration < 30 ng/mL, those with a 25(OH)D concentration ≥ 30 ng/mL had a significantly lower risk of GDM (RR 0.61; 95% CI: 0.43, 0.86), especially those who were overweight before pregnancy (RR 0.32; 95% CI: 0.16, 0.64). The restricted cubic splines model showed an inverted J-shaped relationship between vitamin D concentrations and GDM risk. CONCLUSIONS The risk of GDM was significantly reduced in twin pregnant women with vitamin D concentrations ≥ 30 ng/mL in the second trimester. BACKGROUND ChiCTR-OOC-16,008,203. Retrospectively registered on 1 April 2016.