To assess the status of 25-hydroxyvitamin D [25(OH)D] in pregnant women in their second and third trimesters, who reside and work indoors in Guiyang, China. A total of 311 pregnant women in their 12th to 40th gestational week were engaged in employment located indoors in the urban area of Guiyang and completed a questionnaire on living habits. Levels of serum 25(OH)D were measured from fasting venous blood by liquid chromatography-mass spectrometry (LC-MS/MS). Levels of 25(OH)D were classified as vitamin D deficient [25(OH)D<20 ng/ml], insufficient [20 ng/ml≤25(OH)D<30 ng/ml], or sufficient [25(OH)D≥30 ng/ml]. The mean serum level of 25(OH)D was 14.69±6.81 ng/ml. Vitamin D deficiency, insufficiency and sufficiency were found in 260 (83.6%), 39 (12.5%), and 12 (3.9%) women, respectively. The mean level of 25(OH)D in the third trimester was significantly higher than in the second trimester (p<0.001). The mean 25(OH)D level in summer (June, July, August) was significantly higher than in the other seasons (p<0.001). The 25(OH)D level in pregnant women compliant with pre-natal calcium or multivitamin supplements was higher than in those not taking supplements (p<0.001). These results suggest that pregnant women residing in Guiyang urban area and working indoors are at high risk of vitamin D insufficiency, particularly during spring, winter, and autumn, regardless of use of pre-natal calcium and multivitamins. Appropriate vitamin D supplementation is necessary to improve maternal vitamin D nutrition.