Previous studies indicate reduced risk of type 1 diabetes after intake of vitamin D supplements during pregnancy or early childhood. We aimed to test whether lower maternal serum concentrations of 25-hydroxy-vitamin D (25-OH D) during pregnancy were associated with an increased risk of childhood-onset type 1 diabetes. In this case-control study nested within a cohort of 29,072 women in Norway, 25-OH D levels were measured using a radioimmunoassay on samples from late pregnancy in 109 women delivering a child who developed type 1 diabetes before 15 years of age (case subjects) and from 219 control women. Dividing the levels of maternal 25-OH D into quartiles, there was a trend toward a higher risk of type 1 diabetes with lower levels of vitamin D during pregnancy. The odds of type 1 diabetes was more than twofold higher for the offspring of women with the lowest levels of 25-OH D compared with the offspring of those with levels above the upper quartile. Given future replication in independent cohorts, our findings provide support for the initiation of a randomized intervention trial to prevent type 1 diabetes in children by enhancing maternal 25-OH D status during pregnancy.