The predisposing factors of preeclampsia may endanger the mother's heath as well as her neonate. One hypothesis related to preeclampsia is vitamin D deficiency or insufficiency. This study was conducted to evaluate the relationship between preeclampsia and the serum level of 25-hydroxyvitamin D (25-OH-D) in mothers and their neonates. In this case-control study, we recruited 41 preeclamptic and 50 healthy women from the Imam Khomeini Hospital in Ahvaz, Iran. Venous blood (2 ml) from mothers (in time of labor) and 2 ml of blood from the umbilical cord were taken, centrifuged, stored at -30°C and sent to a laboratory for analysis of 25-OH-D by ELISA. Vitamin D levels<20 ng/ml were regarded as deficiency, levels between 21-29 ng/ml were regarded as insufficiency, and if levels were higher than 30 ng/ml, these were considered normal. Independent t-test, chi-square, Spearman correlation coefficient and logistic regression were used to analyze data. Mean levels of 25-OH-D were significantly lower in preeclamptic women (15.2±13.6 vs. 23.3±15.3 ng/ml, p=0.001) and in their neonates (15.2±13.1 vs. 21.6±12.6 ng/ml, p=0.01) compared to normal pregnant women and their neonates. There was a significant relationship between the levels of vitamin D in preeclamptic women with levels of this vitamin in their neonates (r=0.901, p=0.0001). 25-OH-D deficiency that exist in preeclamptic mothers, may be a health risk for their infants, therefore, early use of vitamin D supplement with higher dose than 400 IU in Iranian women is recommended.