Abstract: Objective To investigate and analyze the levels of serum 25-hydroxy-vitamin D［25-(OH)D］ in 4 013 pregnant women in middle or late stages. Methods A total of 4 013 pregnant women were selected as research objects and all performed detection of serum 25-(OH)D. The serum 25-(OH)D levels and deficiency rates of pregnant women in the second and third trimester of pregnancy were observed, and the correlation between vitamin D intake in the third trimester and serum 25-(OH)D deficiency was analyzed. Results The average levels of serum 25-(OH)D in the second trimester and third trimester were(42.08±15.26)nmol/L and(40.5±15.9)nmol/L, respectively, and their deficiency rates were 65.79% and 73.44%, respectively. There were 1 406(35.04%)pregnant women in third trimester of pregnancy taking vitamin D at a dosage of 400 IU or above daily, 1 057(26.34%)occasionally taking vitamin D, and 1 550(38.62%)taking no vitamin D. Conclusion Vitamin D deficiency probably occurs in more than a half pregnant women in the middle and late pregnancy. The daily nutritional intervention and appropriate vitamin supplementation can improve status of the vitamin D deficiency, and reduce the incidence of related complications during pregnancy.