The purpose of this study was to evaluate the potential of rapeseed proteins including Napin, Cruciferin, and Oleosin as precursors of antimicrobial peptides (AMPs), and to investigate physicochemical properties, secondary structures, toxicity, and allergenicity of AMPs using several bioinformatics tools such as BIOPEP, CAMP, APD, SOPMA, ToxinPred, and AllergenFP. A total of 26 novel AMPs were obtained by in silico hydrolysis using nine proteases, and six peptides were tested positive by all the four algorithms including Random Forest (RF), Support Vector Machines (SVM), Artificial Neural Network (ANN), and Discriminant Analysis (DA). More AMPs were generated from Cruciferin than from Napin and Oleosin. Trypsin was the most effective enzyme for AMPs production compared with other used proteases. About two-third of peptides were cationic. Interestingly, most peptides were extended AMPs. All AMPs were predicted to be non-toxic, and 14 peptides were non-allergenic. These results indicate that rapeseed protein is a good potential source of AMPs as demonstrated by in silico analyses and the theoretical knowledge obtained provides a basis for further development and production of rapeseed AMPs. PRACTICAL APPLICATIONS: Rapeseed protein is a high-quality plant protein resource. However, it is usually used as animal feed or fertilizer. Effective enzymatic hydrolysis of rapeseed protein can release bioactive peptides and improve the utilization value. This study indicates that rapeseed protein is a good potential source of AMPs as demonstrated by in silico analyses. The theoretical knowledge obtained provides a basis for further development and production of rapeseed AMPs.