Plant defensins (PDFs), short peptides with strong antibacterial activity, play important roles in plant growth, development, and stress resistance. However, there are few systematic analyses on in . Here, bioinformatics methods were used to identify genome-wide in , and systematically analyze physicochemical properties, expansion pattern, phylogeny, and expression profiling of under diverse nutrient stresses. A total of 37 full-length homologs, divided into two subgroups ( and ), were identified in the rapeseed genome. A total of two distinct clades were identified in the BnaPDF phylogeny. Clade specific conserved motifs were identified within each clade respectively. Most were proved to undergo powerful purified selection. The members had enriched -elements related to growth and development, hormone response, environmental stress response in their promoter regions. GO annotations indicate that the functional pathways of are mainly involved in cells killing and plant defense responses. In addition, bna-miRNA164 and bna-miRNA172 respectively regulate the expression of their targets and . The expression patterns of were analyzed in different tissues. was mainly expressed in the roots, whereas and were both expressed in stamen, pericarp, silique, and stem. However, the other members showed low expression levels in various tissues. Differential expression of under nitrate limitation, ammonium excess, phosphorus starvation, potassium deficiency, cadmium toxicity, and salt stress indicated that they might participate in different nutrient stress resistance. The genome-wide identification and characterization of will enrich understanding of their molecular characteristics and provide elite gene resources for genetic improvement of rapeseed resistance to nutrient stresses.