A novel antibacterial peptide specific to Streptococcus pyogenes was produced from dried fruit protein of Brucea javanica (L.) Merr. A mixture of active peptides from the fruit protein was produced in vitro by pepsin hydrolysis. The hydrolysate was purified by reverse-phase HPLC, and antimicrobial peptides active against Gram-negative and Gram-positive bacteria were analysed using SDS-PAGE and nanoLC-MS/MS. Here, four possible peptides were obtained and chemically synthesized for comparative study of the growth inhibition of Strep. pyogenes. One chemically synthesized peptide with a molecular mass of 1168·31 Da, His-Thr-Leu-Cys-Met-Asp-Gly-Gly-Ala-Thr-Tyr, showed the most potent antibacterial activity against Strep. pyogenes. This 11-amino acid peptide was named Brucin. Its bacterial inhibitory activity was 16-fold and 12·5-fold higher than penicillin G and chloramphenicol, respectively, with a MIC value of 20 μmol l-1. The results suggest that Brucin, a potent antibiotic peptide, may be developed as an alternative drug for the treatment of the disease caused by Strep. pyogenes. © 2013 The Society for Applied Microbiology.