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[Objectives] The objectives of this study were to identify the defensin-like(DEFL) gene family of pear(Pyrus bretschneideri) genome, and analyze their sequence characteristics, evolutionary relationship, gene structure, conservatively functional domains of amino acid sequences, chromosomal locations and expression characteristics, and provide a reference to the functional research and application of PbrDEFL.[Methods] Based on the whole genome sequencing of P. bretschneideri, the sequences of PbrDEFL were identified and analyzed by bioinformatics. Quantitative real-time PCR(RT-qPCR) and transcriptome data were used to analyze the expression patterns of PbrDEFL from various tissues and different stages of pollen tube development. Subcellular localization was used to analyze the distributions of PbrDEFL38 and PbrDEFL15 in cells. Optimum conditions were established for the production and purification of recombinant PbrDEFL38 and PbrDEFL15 from Escherichia coli.[Results] The results showed that PbrDEFL family were divided into 2 groups, which included 39 DEFL genes(PbrDEFL1-PbrDEFL39) containing the stable cysteine αβ model(CS αβ motif). Members of PbrDEFL gene family were unevenly distributed among 9 chromosomes and purification selection was the driving force of PbrDEFL gene family. RT-qPCR analysis showed that the expression of PbrDEFL presented tissue-specificity, mainly expressed in pollen or leaves; and over 60% of PbrDEFL genes showed a single peak expression trend during the pollen tube development in two pear cultivars('Dangshansuli' and 'Huanghua'). The prediction of PbrDEFL subcellular localization indicated that all proteins were located outside the cell, which was further confirmed by transient expression of PbrDEFL38-GFP and PbrDEFL15-GFP in tobacco. Additionally, the expression and purification conditions of PbrDEFL38 and PbrDEFL15 from E.coli were further analyzed. And the most suitable induction was 15℃ and 0.5 mmol·L-1 IPTG.[Conclusions] This study showed that the members of PbrDEFL gene family were highly conserved, and function was mainly outside the cell. It played an important role in the process of plant immune defense as well as in the pear pollen tube growth and self-incompatibility. [ABSTRACT FROM AUTHOR]