An antifungal protein (GtAFP1) showing antimicrobial activity against phytopathogenic fungi was purified from leaves of Gentiana triflora. The deduced amino acid sequence of the cDNA of the corresponding gene, GtAFP1, showed 94, 75, 72 and 63% amino acid identities with peroxiredoxin Q from Populus balsamifera x P. deltoides subsp. trichocarpa, Sedum lineare, Suaeda maritima and Arabidopsis thaliana, respectively. The GtAFP1 gene is suggested to be present in the genome in one to two copies and was expressed in the leaves, roots and stems. Expression of GtAFP1 was induced by treatment with salicylic acid, but not methyl jasmonate. Recombinant GtAFP1 protein showed not only antifungal activity but also thioredoxin-dependent peroxidase activity. Overexpression of GtAFP1 in tobacco plants improved tolerance not only against fungal diseases but also against oxidative stress. These results indicate that GtAFP1 might act as a disease and oxidative stress defensive gene in plants and could be useful for engineering stress-resistant plants.