Objective Whether vitamin D deficiency in pregnancy is a cause of pre-eclampsia remains controversial. Most previous studies to date have assessed exposure at only one time-point in pregnancy.We assessed longitudinal vitamin D status during pregnancy and the risk of pre-eclampsia.Design Prospective cohort study.Setting Seventeen urban obstetric hospitals, Canada.Population Pregnant women who were participants in a trial of vitamin C and E supplementation for the prevention of preeclampsia. Canadian participants who consented to participate in a biobank with plasma specimens available at the baseline visit were included (n = 697).Methods Maternal plasma 25-hydroxyvitamin D (25(OH)D) concentrations were measured at 12-18 and 24-26 weeks of gestation using chemiluminescence immunoassay.Main outcome measures Pre-eclampsia.Results Of the women, 39% were vitamin D deficient (25(OH)D <50 nmol/l). A strong positive correlation was observed in maternal 25(OH)D concentrations between the two gestational age windows (r = 0.69, P < 0.0001). Mean maternal 25(OH)D concentrations at 24-26 weeks of gestation were significantly lower in women who subsequently developed pre-eclampsia compared with those who did not (mean ± SD: 48.9 ± 16.8 versus 57.0 ± 19.1 nmol/l, P = 0.03). Women with 25(OH)D < 50 nmol/l at 24-26 weeks gestation experienced an increased risk of pre-eclampsia (adjusted odds ratio 3.24, 95% confidence interval 1.37-7.69), whereas the association was not statistically significant for maternal 25(OH)D level at 12-18 weeks of gestation.Conclusions Lower maternal 25(OH)D levels at late mid-trimester were associated with an increased risk of pre-eclampsia.