Abstract: Objective To investigate vitamin D (VD) levels of newborns in Yangzhou, and examine the risk factors of neonatal VD deficiency. Methods The pregnant women who set up health card in Maternal and Child Health Hospital of Yangzhou from May 2014 to May 2015 during the first trimester were selected as the initial study subjects. According to inclusion and exclusion criterion, 3 913 healthy singleton live birth newborns were enrolled in the present study, and related information of pregnant women and newborns were collected with a self-designed questionnaire. The VD concentration of pregnant women and newborns were examined by enzyme-linked immunosorbent assay. The distribution characteristics and risk factors of neonatal VD were described and analyzed. Results The levels of neonatal VD were (28.79±8.37) nmol/L in Yangzhou. The prevalence of sufficiency, insufficiency, and deficiency were 2.2%, 11.7%, and 86.1%, respectively. Multivariate logistic regression model analysis showed that VD deficiency (OR=3.16, 95%CI:2.00-5.00, P<0.001) and VD insufficiency (OR=1.67, 95%CI:1.04-2.67, P=0.033) in the second trimester of pregnancy, VD deficiency (OR=8.64, 95%CI:5.64-13.24, P<0.001) and VD insufficiency (OR=1.63, 95%CI:1.07-2.49, P=0.024) in the third trimester of pregnancy, and fetal delivery in winter-spring (OR=1.49, 95%CI:1.13-1.97, P=0.004) were risk factors of neonatal VD deficiency. Conclusions VD deficiency of newborns were common in Yangzhou. The VD status of pregnant women in the second and third trimesters of pregnancy and the season of fetal delivery were associated with neonatal VD deficiency.