A novel plant hairpin-like defense polypeptide named EcAMP3 was isolated from latent barnyard grass (Echinochloa crusgalli L.) seeds. The native peptide and its recombinant analogue were characterized. EcAMP3 displays antifungal and antibacterial activity in vitro. The gene family encoding EcAMPs precursor protein was also characterized; the genes and pseudogenes of this family show 97-100% homology. Every member of EcAMPs precursor family contains seven identical cysteine motifs: C1XXXC2(11-13)C3XXXC4. One of those motifs corresponds to the isolated peptide. EcAMP3 is the first member of the plant hairpin-like peptide family that inhibits the growth of phytopathogenic bacteria. Obtained results can explain the nature of the complex resistance of barnyard grass to a variety of pathogenic microorganisms. © 2013 Elsevier Masson SAS. All rights reserved.