AbstractContext:Vitamin D insufficiency is associated with increased cardiovascular events in the general population. Additionally, low serum 25-hydroxyvitamin D [25(OH)D] is associated with endothelial dysfunction and arterial stiffness. However, little is known about the association between serum 25(OH)D level and myocardial blood flow.Objective:Our objective was to examine the association between serum 25(OH)D levels and coronary flow reserve (CFR) measured by 13N-positron emission tomography in asymptomatic middle-aged male twins.Design:The Emory Twin Study is a cross-sectional study of soldiers from the Vietnam Era Registry.Setting:The study was conducted at the General Clinical Research Center, Emory University, Atlanta, GA.Participants:A total of 368 middle-aged male twins were enrolled for the study. Serum 25(OH)D levels were measured in all subjects and classified as vitamin D insufficiency [25(OH)D <30 ng/ml] or sufficiency [25(OH)D ≥30 ng/ml]. Positron emission tomography with [13N]ammonia was used to evaluate myocardial blood flow at rest and after adenosine stress. CFR was measured as the ratio of maximum to rest myocardial blood flow.Main Outcome Measure:Primary outcome was CFR measurement.Results:Mean overall serum 25(OH)D concentration was 37.0 ± 21.4 ng/ml; 167 twins (45%) were vitamin D insufficient. CFR was significantly lower in subjects with vitamin D insufficiency compared with subjects with vitamin D sufficiency (2.41 vs. 2.64; P = 0.007), even after adjustment for traditional cardiovascular risk factors, serum PTH, calcium, and phosphorus levels, and season. An abnormal CFR (CFR <2) was more prevalent in subjects with vitamin D insufficiency than with vitamin D sufficiency (31 vs. 20%; P = 0.03). In addition, in vitamin D status-discordant twin pairs, CFR was significantly lower in the vitamin D-insufficient twin than in the vitamin D-sufficient co-twin (2.35 vs. 2.58; P = 0.037).Conclusion:Vitamin D insufficiency is associated with lower CFR in men. This association may help explain some of the increased cardiovascular risk reported in individuals with vitamin D insufficiency.