Lamprey was considered to be one of the most basal jawless vertebrate representatives for studying vertebrate evolution, embryo development, and the origin of adaptive immunity. Here we investigated the effect of the gut-derived Aeromonas on the lamprey leukocytes proteome using the label-free liquid chromatography-tandem mass spectrometry for quantitative proteomics analysis. Significant difference was observed in the regulation of 34 out of 755 proteins in Aeromonas-immunized lamprey. 31 proteins were only identified in saline solution-immunized lamprey and 47 proteins were only identified in Aeromonas-immunized lamprey. Quantitative real-time polymerase chain reaction was used to validate the results of the proteomic analysis. The differentially expressed proteins were found to be associated with several different biological processes. The identification of leukocytes proteins essential for lamprey adaptive immune response induced by gut-derived Aeromonas strain could supply important information on lamprey-Aeromonas interactions and VLR-based adaptive immune signal pathways.