Defensins of birds belong to the large family of antimicrobial peptides that are key components of mucosal innate immunity. Among them, avian defensin 2 (AvBD2) has been pointed out for its antimicrobial activity more efficient against Gram+ than against Gram - bacterial strains, as determined by radial diffusion assay . To determine structural features that may be involved in the mode of action of AvBD2 and in its peculiar antibacterial spectrum, synthetic form of AvBD2 was prepared and characterized structurally by NMR 131. The beta-sheet 3D-structure of AvBD2, typical of beta-defensins, revealed a protruding K31 residue in a hydrophobic environment of the C-terminal part of the molecule, which may interact preferentially with bacterial surfaces. Synthetic enantiomers and point-mutated (K31A) variants of AvBD2, either folded or linear, were then synthesised and assayed on various bacterial strains. Both L- and D-AvBD2 exhibited similar antimicrobial activity, indicating that the bacterial molecular target is not chiral. The loss of the folding increased importantly the minimum inhibitory concentration of the defensin, in a more marked way towards Gram+ than towards Gram- bacterial species. Finally the point mutation of the K31A AvBD2 also reduced significantly the antibacterial activity. Taken together, the results reveal a different impact of structural features on antibacterial activity of this antimicrobial peptide according to the bacterial type, suggesting that the antimicrobial mechanism may differ between Gram+ and Gram- bacterial species.