Summary. CD47 (integrin-associated protein) serves as a receptor for thrombospondin-1 (TSP-1) and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), and the TSP-1/CD47 interaction has been believed to augment integrin-mediated platelet function. Here, employing SHPS-1-immunoglobulin (Ig) as a ligand, we have newly demonstrated that CD47 acts as an inhibitory receptor for platelet function. The binding of SHPS-1-Ig was solely mediated by CD47, because CD47-deficient platelets failed to bind murine SHPS-1-Ig. The human SHPS-1/CD47 interaction inhibited the platelet aggregation induced by several kinds of agonists at a low concentration. Moreover, human SHPS-1 expressed on the cell surface as well as soluble SHPS-1-Ig markedly inhibited the platelet spreading on, but not initial adhesion to, immobilized fibrinogen. Again, neither murine SHPS-1 expressed on the cell surface nor murine SHPS-1-Ig inhibited the spreading of CD47-deficient platelets. We further investigated the tyrosine phosphorylation of signaling proteins during platelet spreading on immobilized fibrinogen. Unexpectedly, SHPS-1 inhibited αIIbβ3-mediated platelet spreading without disturbing focal adhesion kinase (FAK) tyrosine phosphorylation. Further examination revealed that SHPS-1 inhibited the tyrosine phosphorylation of α-actinin, a downstream effector of FAK, but not of cortactin. Thus, it is likely that the SHPS-1/CD47 interaction inhibits αIIbβ3-mediated outside-in signaling by interfering with the downstream pathway of FAK. Taken together, our data suggest that SHPS-1 negatively regulates platelet function via CD47, especially αIIbβ3-mediated outside-in signaling.