Objectives— α-Defensins are natural antibiotics made by neutrophils that have been reported to modulate cholesterol metabolism and vascular function; however, their role in vivo remains largely unknown. We hypothesized that α-defensins 1 to 3 (DEFA1–3) are associated with serum lipids and vascular reactivity in humans. Methods and Results— One hundred thirteen apparently-healthy White men, participants in a prospective study of cardiovascular risk factors, were assessed for a lipid profile, insulin sensitivity ( S I , frequently-sampled intravenous glucose tolerance test), and non-stressed circulating DEFA1–3 (ELISA). In a subset of 52 subjects, vascular reactivity (high-resolution ultrasound of the brachial artery) was also assessed. Subjects in the highest quartile for plasma DEFA1–3 were found to be leaner and more insulin sensitive, and to have significantly reduced total and LDL-cholesterol, compared with subjects in the lowest quartile for circulating DEFA1–3 ( P <0.0001 to P =0.002 for linear trend ANOVA). The associations with serum lipids persisted after adjustment for age, body mass index, insulin sensitivity, and smoking (which was associated with reduced plasma DEFA1–3 concentrations). Finally, endothelium-independent vasodilation increased with increasing circulating DEFA1–3 ( P =0.003) and this association was not explained by age, body mass index, serum cholesterol, insulin sensitivity, or smoking. Conclusions— Circulating DEFA1–3 are associated with serum cholesterol and vascular reactivity in humans. α-Defensins may have clinical implications in patients with either hypercholesterolemia or vascular dysfunction.