Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. In recent years, a series of outbreaks of serotype A FMD have occurred in many countries. High-affinity neutralizing antibodies against a conserved epitope have the potential to provide protective immunity against diverse subtypes of FMDV serotype A and to protect against future pandemics. In this study, we produced an A serotype FMDV-specific monoclonal antibody (MAb) against the viral capsid protein VP1, designated 9A9, that potently neutralized FMDV A/JLYS/CHA/2014 with a 50 % neutralization titer (NT50) of 4,096. GST-fusion proteins expressing truncated peptides of VP1 were subjected to Western blot analysis using MAb 9A9, and it was found that the peptide (143)RGDLGPLAARL(153) of VP1 was the minimal epitope for MAb 9A9 binding. Western blot analysis also revealed that the epitope peptide could be recognized by positive sera from serotype A FMDV-infected pigs and cattle. Subsequent alanine-scanning mutagenesis analysis revealed that residues Gly(147) and Leu(149) of the 9A9-recognized epitope are crucial for MAb 9A9 binding. Furthermore, under immunological pressure selected by MAb 9A9, a single amino acid residue replacement (L149P) occurred in a viral neutralization-escape mutant, which verified the location of a critical residue of this epitope at Leu(149). Importantly, the epitope (143)RGDLGPLAARL(153) was highly conserved among different topotypes of serotype A FMDV strains in sequence alignment analysis. Thus, the results of this study could have application potential in the development of epitope-based vaccines and a suitable MAb-based diagnostic method for detection of type A FMDV as well as quantitation of antibodies against FMDV serotype A.