Maternal vitamin D deficiency causes pregnancy complications and delayed skeletal development in offspring. This study aimed at identifying demographic and lifestyle factors associated with vitamin D status in pregnant Japanese women. A total of 284 healthy pregnant women in the second trimester were recruited at a university hospital in Tokyo, between June 2010 and July 2011. Serum 25-hydroxyvitamin D (25(OH)D) concentrations were measured using chemiluminescent immunoassay. We assessed vitamin D intake using a self-administered diet history questionnaire and asked participants about lifestyle variables, including daily duration of sunlight exposure and supplement use. The mean (SD) serum 25(OH)D concentration was 9.8 (4.7) ng/mL. Almost 60% of the participants had severe vitamin D deficiency (measured as 25(OH)D<10 ng/mL). Multiple regression analysis showed that multigravidity, pre-pregnancy non-underweight status, higher energy-adjusted vitamin D intake, and use of vitamin D supplements were correlated with higher serum 25(OH)D concentrations (β=0.245, β=−0.119, β=0.226, and β=0.197, respectively). In the summer investigation, women with longer durations of sunlight exposure had significantly higher serum 25(OH)D concentrations (β=0.201) that were unrelated to the factors outlined previously. In the winter investigation, women with a high education level had higher serum 25(OH)D concentrations than others (β=0.330). Our results would be useful for identifying pregnant women at a high risk of low vitamin D status, such as primigravidae and those with pre-pregnancy underweight status, low education level, low vitamin D intake, and short durations of sunlight exposure.