Objective·To investigate the reference range for serum 25-hydroxyvitamin D in pregnant women in Shanghai. Methods·A total of 4969 healthy pregnant women in second trimester were enrolled, in whom there were 554 women aged over or equal to 35 years old and 4415 women aged under 35 years old. Meanwhile, 1048 non-pregnant women in child-bearing period were enrolled. The new born children had no complications of vitamin D deficiency such as rickets. The levels of 25-hydroxyvitamin D of all participants were detected by ELISA. The reference interval was defined as the central 95% range between the 2.5th and the 97.5th percentiles. Results·The 95% reference range for 25-hydroxyvitamin D levels showed a positive skewness distribution in second trimester pregnant women. The median of 25-hydroxyvitamin D levels was 36.8 nmol/L. The maximum was 108.6 nmol/L whereas the minimum was 11.8 nmol/L. The 95% reference range for 25-hydroxyvitamin D levels was from 15.9 nmol/L to 70.3 nmol/L. The median 25-hydroxyvitamin D was 36.6 nmol/L in women less than 35 years old, and 35.5 nmol/L in women aged over or equal to 35 years old. A significant difference was found between women aged under 35 years old and women aged over or equal to 35 years old (Z=-2.75, P<0.05). The median, maximum and minimum of 25-hydroxyvitamin D in non-pregnant women was 38.5 nmol/L, 110.5 nmol/L, 12.1 nmol/L, respectively, which were all higher than those in pregnant women (P<0.05). Conclusion·There was a lack of vitamin D in pregnant women in Shanghai, especially in pregnant women aged over or equal to 35 years old, despite the multivitamin supplements. However, no complications of vitamin D deficiency such as rickets were found in new born children. To determine the reference range of vitamin D in Shanghai is helpful for pregnancy women in taking vitamin supplement, especially important in preventing maternal and fetal complications caused by vitamin D deficiency.