Objective Neointima formation is associated with stenosis and subsequent thrombosis in arteriovenous grafts (AVGs). A role of integrin 3 in the neointima formation of AVGs remains poorly understood. Approach and Results In integrin 3(-/-) mice, we found significantly accelerated occlusion of AVGs compared with the wild-type mice. This is caused by the development of neointima and lack of endothelial regeneration. The latter is a direct consequence of impaired functions of circulating angiogenic cells (CACs) and platelets in integrin 3(-/-) mice. Evidence suggests the involvement of platelet regulating CAC homing to and differentiation at graft sites via transforming growth factor-1 and Notch signaling pathway. First, CACs deficient of integrin 3 impaired adhesion activity toward exposed subendothelium. Second, platelets from integrin 3(-/-) mice failed to sufficiently stimulate CACs to differentiate into mature endothelial cells. Finally, we found that transforming growth factor-1 level was increased in platelets from integrin 3(-/-) mice and resulted in enhanced Notch1 activation in CACs in AVGs. These results demonstrate that integrin 3 is critical for endothelial cell homing and differentiation. The increased transforming growth factor-1 and Notch1 signaling mediates integrin 3(-/-)-induced AVG occlusion. This accelerated occlusion of AVGs was reversed in integrin 3(-/-) mice transplanted with the bone marrow from wild-type mice. Conclusions Our results suggest that boosting integrin 3 function in the endothelial cells and platelets could prevent neointima and thrombosis in AVGs.