IMPORTANCE: Vitamin D deficiency has been associated with hypertension, diabetes mellitus, and incident stroke. Little is known about the association between vitamin D and subclinical cerebrovascular disease. OBJECTIVE: To examine the relationship of 25-hydroxyvitamin D (25[OH]D) levels with cerebrovascular abnormalities as assessed on brain magnetic resonance imaging (MRI) among participants of the Atherosclerosis Risk in Communities (ARIC) Brain MRI study. DESIGN, SETTING, AND PARTICIPANTS: Participantswere white and black adults aged 55 to 72 years with no history of clinical stroke who underwent a cerebral MRI at ARIC visit 3 (n = 1622) and a second cerebral MRI approximately 10 years later (n = 888). EXPOSURES: The 25(OH)D level was measured by mass spectrometry at visit 3, with levels adjusted for calendar month and categorized using race-specific quartiles. MAIN OUTCOMES AND MEASURES: The cross-sectional and prospective associations of 25(OH)D levels with white matter hyperintensities (WMHs) and MRI-defined infarcts were investigated using multivariable regression models. RESULTS: The mean age of the participants was 62 years, 59.6%were women, and 48.6% were black. Lower 25(OH) D levels were not significantly associated with WMH score of severity, prevalent high-grade WMH score (≥3), or prevalent infarcts in cross-sectional, multivariable-adjusted models (all P > .05). Similarly, no significant prospective associations were found for lower 25(OH)D levels with change in WMH volume, incident high WMH score (≥3), or incident infarcts on the follow-up MRI, which occurred approximately 10 years later. CONCLUSIONS AND RELEVANCE: A single measure of 25(OH)D was not cross-sectionally associated with WMH grade or prevalent subclinical infarcts and was not prospectively associated with WMH progression or subclinical brain infarcts seen on serial cerebral MRIs obtained approximately 10 years apart. These findings do not support optimizing vitamin D levels for brain health.