Abstract: TMT labeling, high performance liquid chromatography (HPLC) and quantitative proteomics based on mass spectrometry were used to investigate the differential expression proteins (DEPs) in spleen of pufferfish (Takifugu obscurus) infected with Aeromonas hydrophila for 12 h. The spleens of the experimental group and the control group were collected at 12 h after infection, then the expression level of proteins was detected and DEPs were screened through identification and quantification analysis. In addition, DEPs were further analyzed with subcellular localization, gene oncology(GO) and kyoto encyclopedia of genes and genomes(KEGG). A total of 306 DEPs were screened, among which 120 proteins were up-regulated and 186 proteins were down-regulated. Four (1 up-regulated, 3 down-regulated) immune-related proteins were chosen for validation with parallel response assay (PRM), and the results were consistent with those of the quantitative proteomics. The results of subcellular localization showed that DEPs were mainly located in cytoplasm, nucleus and extracellular. GO analysis found that the main biological processes involved in DEPs were metabiolic processes, cellular processes and single biological processes, and the main molecular functions were binding, catalytic activity and mechanism analysis activity. Bioinformatics analysis indicated that DEPs were mainly associated with ribosome, retinol metabolism, porphyrin and chlorophyll metabolism, ECM-receptor interaction, phagosome, folate biosynthesis, drug metabolism, tyrosine metabolism and so on. The potential mechanism of regulated proteins by A. hydrophila infection in spleen pathology needs further investigation.