Hypercholesterolemia is associated with phenotypic changes in endothelial cell function that lead to a pro-inflammatory and prothrombogenic state in different segments of the microvasculature. CD40 ligand (CD40L) and its receptor CD40 are ubiquitously expressed, and mediate inflammatory responses and platelet activation. The objective of this study was to determine whether CD40/CD40L, in particular T-cell CD40L, contributes to microvascular dysfunction induced by hypercholesterolemia. Intravital microscopy was used to quantify blood cell adhesion in cremasteric postcapillary venules, endothelium-dependent vasodilation responses in arterioles, and microvascular oxidative stress in wild-type (WT) C57Bl/6 mice, CD40-deficient (^-/-), CD40L^-/- or lymphocyte-deficient (SCID) mice placed on a normal (ND) or high-cholesterol (HC) diet for 2 wks. WT-HC mice exhibited an exaggerated leukocyte and platelet recruitment in venules, and impaired vasodilation responses in arterioles when compared to ND counterparts. Deficiency of CD40, CD40L or lymphocytes attenuated these responses to HC. The HC phenotype was rescued in CD40L^-/- and SCID mice by transfer of WT T-cells. Bone marrow chimeras revealed roles for both vascular- and blood cell-derived CD40 and CD40L in the HC-induced vascular responses. Hypercholesterolemia induced an oxidative stress in both arterioles and venules of WT mice, which was abrogated by either CD40 or CD40L deficiency. Transfer of WT T-cells into CD40L^-/- mice restored the oxidative stress. These results implicate CD40/CD40L interactions between circulating cells and the vascular wall in both the arteriolar and venular dysfunction elicited by hypercholesterolemia, and identify T-cell associated CD40L as a key mediator of these responses.