Royal jelly (RJ) from honeybees is known to be composed of diverse antibacterial components with broad-spectrum antibacterial activity. In this study, a model organism of bacteria (Escherichia coli) was challenged with RJ and the proteomic changes were investigated. A total of 209 differentially expressed proteins (DEPs) were identified; 200 (95.70%) DEPs were upregulated and 9 (4.30%) were downregulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied.