The relationship between vitamin D metabolites and subclinical vascular disease is controversial. Because low serum levels of 25-hydroxyvitamin D (25(OH)D) have been associated with many cardiovascular disease (CVD) risk factors, we hypothesized that serum 25(OH)D levels would be inversely associated with inflammation as measured by C-reactive protein (CRP) and with subclinical vascular disease as measured by carotid intimal medial thickness (cIMT) and coronary artery calcification (CAC). We measured 25(OH)D levels in 650 Amish participants. CAC was measured by computed tomography and cIMT by ultrasound. The associations of 25(OH)D levels with natural log(CAC + 1), cIMT, and natural log(CRP) levels were estimated after adjustment for age, sex, family structure, and season of examination. Additional analyses were carried out adjusting for body mass index (BMI) and other CVD risk factors. 25(OH)D deficiency (<20 ng/ml) and insufficiency (21-30 ng/ml) were common among the Amish (38.2% and 47.7%, respectively). 25(OH)D levels were associated with season, age, BMI, and parathyroid hormone levels. In neither the minimally or fully adjusted analyses were significant correlations observed between 25(OH)D levels and CAC, cIMT, or CRP (R (2) < 0.01 for all). Contrary to our hypothesis, this study failed to detect a cross-sectional association between serum 25(OH)D levels and CAC, cIMT, or CRP. Either there is no causal relationship between 25(OH)D and CVD risk, or if there is, it may be mediated through mechanisms other than subclinical vascular disease severity.