Aspirin is common preventative therapy in patients at risk for cardiovascular diseases, yet little is known about how aspirin protects the vasculature in hypercholesterolemia. The present study determines whether aspirin, NO-releasing aspirin (NCX-4016), a selective COX-1 inhibitor (SC560), or genetic deficiency of COX-1 prevents the inflammatory and prothrombogenic phenotype assumed by hypercholesterolemic (HC) venules. Aspirin or NCX-4016 (60 mg/kg) was administered orally for the last wk of a 2 wk HC diet. COX-1 deficient (COX-1^-/-) and wild type (WT) mice were transplanted with WT (WT/COX^-1-/-) or COX-1^-/- (COX-1^-/-/WT) bone marrow, respectively. HC-induced adhesion of platelets and leukocytes in murine intestinal venules, observed with intravital fluorescence microscopy, was greatly attenuated in aspirin-treated mice. Adhesion of aspirin-treated platelets in HC venules was comparable to untreated platelets, while adhesion of SC560-treated platelets was significantly attenuated. HC-induced leukocyte and platelet adhesion in COX-1^-/-/WT chimeras were comparable to SC560-treated mice, while the largest reductions in blood cell adhesion were in WT/COX-1^-/- chimeras. NCX-4016 treatment of platelet recipients or donors attenuated leukocyte and platelet adhesion independent of platelet COX-1 inhibition. Platelet- and endothelial cell-associated COX-1 promote microvascular inflammation and thrombogenesis during hypercholesterolemia, yet NO-releasing aspirin directly inhibits platelets independent of COX-1.