Objective: To analyze the level and clinical significance of serum 25-hydroxyvitamin D (25-OH-D) levels in pregnant women with subclinical hypothyroidism (SCH) and gestational diabetes mellitus (GDM). Method: One hundred pregnant women of 24-28 weeks' pregnancy with SCH combined GDM were selected into the observation group, and 100 healthy pregnant women were selected into the control group during the corresponding period. Examined the thyroid stimulating hormone (TSH), free thyroxine (FT4), 25-OH-D, serum calcium (Ca2+), fasting plasma glucose (FPG), and glycosylated hemoglobin (HbAlc) levels and thyroid peroxide antibody (TPOAb), Thyroglobulin antibody (TgAb) status. Examine and compare TSH, FT4, Ca2+, FPG, HbAlc, TPOAb, and TgAb at different levels of serum 25-OH-D in the observation group. Compared the 25-0H-D levels and the ratio of different contents of 25-OH-D of TPOAb-positive and TgAbpositive SCH pregnant women. Result: The levels of TSH, FPG and HbAlc in the observation group were significantly higher than those in the control group (P<0.01). Through comparison of FT4 levels between the two groups, the difference had no statistical significance (P>0.05). The levels of 25-OH-D and Ca2+ in the observation group was significantly lower than those in the control group, and the difference had statistical significance (P<0.01). Through comparison of TSH, Ca2+, FPG and HbAlc in groups with different serum 25-OH-D levels, the difference had statistical significance (P<0.01). The positive rates of TPOAb and TgAb of pregnant women in the observation group were higher than in the control group (P<0.05). The vitamin D deficiency rate of TPOAb or TgAb positive pregnant women in the observation group was higher than those in the TPOAb or TgAb negative pregnant women, the difference had statistical significance (P < 0.05). Conclusion: blood glucose level in pregnant women with GDM and SCH increased significantly, blood calcium level decreased significantly. This group of people are more likely to have VitD deficiency. Thyroid stimulating hormone and blood glucose levels in pregnant women are negatively correlated with VitD levels. Therefore, serum 25-OH-D level can be used as an important reference index for gestational diabetes mellitus with subclinical hypothyroidism, and it has great clinical significance to maintain it at a normal level.