Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.