Monoclonal antibodies (mAbs) have provided valuable information on the structure and function of platelet αIIbβ3. Protein disulfide isomerase (PDI) has been implicated in αIIbβ3 activation and binds to thrombin-activated αIIbβ3. Using human platelets as immunogen, we identified a new mAb (R21D10) that inhibits the binding of PDI to platelets activated with a thrombin receptor-activating peptide (T6). R21D10 also partially inhibits T6-induced fibrinogen and PAC-1 binding to platelets, as well as T6- and ADP-induced platelet aggregation. Mutual competition experiments show that R21D10 does not inhibit binding of mAbs 10E5 (anti-αIIb cap domain) or 7E3 (anti-β3 β-I domain) and immunoblot studies indicate that R21D10 binds to β3. Dissociation of αIIbβ3 by EDTA had minimal effect on R21D10 binding. Cryo-electron microscopy of the αIIbβ3-R21D10 Fab complex reveals that R21D10 binds to the β3 I-EGF1 domain and traps an intermediate conformation of αIIbβ3 with semi-extended leg domains. Binding of R21D10 produces a major structural change in the β3 I-EGF2 domain associated with a new interaction between the β3 I-EGF2 and the αIIb thigh domains, which may prevent the swing-out motion of the β3 hybrid domain required for high-affinity ligand binding and protect αIIbβ3 from EDTA-induced dissociation. R21D10 partially reverses the ligand binding priming effect of eptifibatide, suggesting that it can convert the swung-out conformation into the semi-extended conformation. We conclude that R21D10 inhibits ligand binding to αIIbβ3 via a unique allosteric mechanism, which may or may not be related to its inhibition of PDI binding.