The antibiotics resistance phenomena of Aeromonas hydrophila has become serious economic and public health problems for the world aquaculture industry and human health care. In this study, to investigate the instinct antibiotics adaptive mechanism of this pathogen, iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) based quantitative proteomics technologies were performed to compare the differential expression of A. hydrophila in planktonic status in response to chlortetracycline (CTC) stress and then identified total 1552 proteins including 285 altered proteins with 90 increasing and 195 decreasing abundance proteins. The following bioinformatics analysis showed that many metabolic metabolism pathways such as carbon metabolism, pyruvate metabolism, and glycolysis/gluconeogenesis were trend to down-regulated whereas beta-Lactam resistance, RNA degradation, and amino acids biosynthesis processes were more likely to increase in CTC stress. The related pyruvate metabolism and beta-Lactam resistance processes in mRNA level were further measured using the q-PCR method. Thus, an understanding of the behaviors of A. hydrophila in response to CTC would be helpful to reveal the antibiotics adaptive mechanism and for the development of novel antibiotics therapy. [GRAPHCIS].