Junctional adhesion molecule-A (JAM-A) serves as a serotype-independent receptor for mammalian orthoreoviruses (reoviruses). The membrane-distal immunoglobulin-like D1 domain of JAM-A is required for homodimerization and binding to reovirus attachment protein sigma1. We employed a structure-guided mutational analysis of the JAM-A dimer interface to identify determinants of reovirus binding. We purified mutant JAM-A ectodomains for solution-phase and surface plasmon resonance binding studies and expressed mutant forms of full-length JAM-A in Chinese hamster ovary cells to assess reovirus binding and infectivity. Mutation of residues in the JAM-A dimer interface that participate in salt-bridge or hydrogen-bond interactions with apposing JAM-A monomers abolishes the capacity of JAM-A to form dimers. JAM-A mutants incapable of dimer formation form complexes with the sigma1 head that are indistinguishable from wild-type JAM-A-sigma1 head complexes, indicating that sigma1 binds to JAM-A monomers. Residues Glu(61) and Lys(63) of beta-strand C and Leu(72) of beta-strand C' in the dimer interface are required for efficient JAM-A engagement of strain type 3 Dearing sigma1. Mutation of neighboring residues alters the kinetics of the sigma1-JAM-A binding interaction. Prototype reovirus strains type 1 Lang and type 2 Jones share similar, although not identical, binding requirements with type 3 Dearing. These results indicate that reovirus engages JAM-A monomers via residues found mainly on beta-strands C and C' of the dimer interface and raise the possibility that the distinct disease phenotypes produced in mice following infection with different strains of reovirus are in part attributable to differences in contacts with JAM-A.