Herein the cloning and characterization of a defensin gene (PpDfn1) from a cDNA library made from peach (Prunus persica[L.] Batsch) winter bark tissues is described. A partial clone obtained from the library was extended to full length by 5′ Rapid Amplification of cDNA Ends (RACE). The open reading frame of 237 bp codes for a 79 amino acid peptide related to the defensin family of proteins. Sequence comparison of the encoded protein using blast analysis revealed significant homology to defensins from other plant species. RNA gel blot analysis indicated that the gene is seasonally expressed in bark tissues of 1-year-old shoots, and is also expressed in early fruit development. Results of quantitative RT-PCR and protein blot analysis were similar to those of RNA gel blot analyses for the bark tissues. A recombinant version, rDFN1 was expressed in the yeast, Pichia pastoris. It was found that rDFN1 inhibited germination of the fungal pathogens Penicillium expansum and Botrytis cinerea, but not the Gram-negative bacterium Erwinia amylovora. The potential physiological role of PpDFN1 and its antimicrobial properties are discussed.