Background and Purpose- Recent findings suggest that vitamin D, a neuroprotective prohormone, is involved in the pathogenesis of cardiovascular disease. However, previous studies investigating the association between vitamin D and stroke have shown inconsistent findings. In view of these discrepancies, we determined the association of vitamin D status with stroke using data from a population-based study. Methods- Within the RS (Rotterdam Study), an ongoing prospective population-based study, we measured serum 25-hydroxyvitamin D concentrations between 1997 and 2008 in 9680 participants (56.8% women) aged >= 45 years. We assessed a history of stroke at baseline and subsequently followed for incident stroke until January 1, 2016. Regression models were used to investigate the association of serum 25-hydroxyvitamin D with prevalent and incident stroke separately, adjusted for age, sex, study cohort, season of blood sampling, and other cardiovascular risk factors. Results- Of 9680 participants, 339 had a history of stroke at baseline. Serum 25-hydroxyvitamin D concentration was associated with prevalent stroke, adjusted odds ratio per SD decrease, 1.31; 95% CI, 1.14-1.51. After excluding participants with prevalent stroke, we followed 9338 participants for a total of 98 529 person-years. During follow-up, 735 participants developed a stroke. Lower serum 25-hydroxyvitamin D concentration was not associated with a higher stroke risk, adjusted hazard ratio per SD decrease, 1.06; 95% CI, 0.97-1.16. However, severe vitamin D deficiency did show a significant association: hazard ratio, 1.25; 95% CI, 1.05-1.50. Conclusions- In this population-based cohort, we found an association between vitamin D and prevalent stroke. Only severe vitamin D deficiency was associated with incident stroke. This suggests that lower vitamin D levels do not lead to a higher stroke risk but are instead a consequence of stroke.