Lamprey, one of the most basal jawless vertebrate, is an excellent model for studying vertebrate evolution, embryo development, and the origin of adaptive immunity. This study investigated the differentially expressed proteins in lamprey leukocytes in response to the co-stimulation of intestinal Aeromonas and Shewanella by using quantitative proteomics techniques. Significant differentially expressed proteins were identified. Gene Ontology annotation and the Kyoto Encyclopedia of Genes and Genomes pathway based on the significant differentially expressed proteins were analyzed. Most of the differentially expressed proteins were predicted to be involved in important signaling pathways. Quantitative real-time polymerase chain reaction was used to verify the expression of differentially expressed proteins at the mRNA level. The expression of some differentially expressed proteins was not consistent at the mRNA and protein levels. Differentially expressed proteins that are essential for lamprey-intestinal bacteria interaction should be identified to understand the lamprey adaptive immune response induced by gut microbiota.