Objective · To investigate the reference range for serum 25-hydroxyvitamin D in pregnant women in Shanghai. Methods · A total of 4 969 healthy pregnant women in second trimester were enrolled, in whom there were 554 women aged over or equal to 35 years old and 4 415 women aged under 35 years old. Meanwhile, 1 048 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.