Background: To our knowledge, no prospective study has examined the association between vitamin D and cognitive decline or dementia. Methods: We determined whether low levels of serum 25-hydroxyvitamin D (25[OH] D) were associated with an increased risk of substantial cognitive decline in the InCHIANTI population-based study conducted in Italy between 1998 and 2006 with follow-up assessments every 3 years. A total of 858 adults 65 years or older completed interviews, cognitive assessments, and medical examinations and provided blood samples. Cognitive decline was assessed using the Mini-Mental State Examination (MMSE), and substantial decline was defined as 3 or more points. The Trail-Making Tests A and B were also used, and substantial decline was defined as the worst 10% of the distribution of decline or as discontinued testing. Results: The multivariate adjusted relative risk (95% confidence interval [CI]) of substantial cognitive decline on the MMSE in participants who were severely serum 25 (OH) D deficient (levels = 75 nmol/L) was 1.60 (95% CI, 1.19-2.00). Multivariate adjusted random-effects models demonstrated that the scores of participants who were severely 25(OH) D deficient declined by an additional 0.3 MMSE points per year more than those with sufficient levels of 25(OH) D. The relative risk for substantial decline on Trail-Making Test B was 1.31 (95% CI, 1.03-1.51) among those who were severely 25(OH) D deficient compared with those with sufficient levels of 25(OH) D. No significant association was observed for Trail-Making Test A. Conclusion: Low levels of vitamin D were associated with substantial cognitive decline in the elderly population studied over a 6-year period, which raises important new possibilities for treatment and prevention.