Saffold virus (SAFV) is the human Cardiovirus closely related to the Theiler murine encephalomyelitis virus (TMEV), of the family picornaviridae (1). It was reported that, SAFV might cause respiratory, gastrointestinal, and central nervous system infections (1,2). To date 11 genotypes of SAFV have been identified (1, 3). In the present study, the three-dimensional structure of SAFV-3 has been determined at 2.5-Å resolution. Although the architecture of the major capsid proteins VP1, VP2 and VP3 of SAFV-3 is similar to other cardioviruses, there are some differences on the surface loops. The presence of disulphide bond on the surface of VP3, surprisingly diminish the stability and infectivity of SAFV-3. Several capsid-binding and replication inhibitors of other picornaviruses fail to have any effect on SAFV-3. It was also shown that SAFV-3 dissociates in to pentameric subunits upon the genome release.