Serum 25(OH)D concentration has been identified as a possible modifiable risk factor for cardiovascular disease (CVD). We hypothesized that serum 25(OH)D concentration would be associated with calcifications of the left-sided heart valves, which are markers of CVD risk. Aortic Valve Calcium (AVC) and Mitral Annular Calcium (MAC) were quantified from cardiac CT scans performed on 5,530 MESA participants at the baseline exam (2000–2002) and at a follow-up visit at either Exam 2 (2002–2004) or Exam 3 (2004–2005). 25(OH)D was measured from serum samples collected at the baseline exam. Using relative risk regression, we evaluated the multivariable-adjusted risk of prevalent and incident AVC and MAC in this ethnically diverse population free of clinical CVD at baseline. The mean age of participants was 62±10 years; 53% were women, 40% white, 26% black, 21% Hispanic and 12% Chinese. Prevalent AVC and MAC were observed in 12% and 9%, respectively. There were no significant associations between 25(OH)D and prevalent AVC or MAC. Over a mean follow-up of 2.5 years, 4% developed incident AVC and 5% developed incident MAC. After adjusting for demographic variables, each 10 ng/ml higher serum 25(OH)D was associated with a 15% [RR=0.85 (95% CI 0.74, 0.98)] lower risk of incident MAC but not AVC. However, this association was no longer significant after adjusting for lifestyle and CVD risk factors. Results suggest a possible link between serum 25(OH)D and the risk for incident MAC, but future studies with longer follow-up are needed to further test this association.