Aeromonas hydrophila is one of the most common pathogenic bacteria in the aquaculture industry. Despite reports of drug-resistant strains of A. hydrophila in many studies, the internal adaptation mechanism to antibiotics remains unclear. This study used quantitative proteomics to compare the differences in protein expression of A. hydrophila with and without streptomycin (SM) stress for further analysis. A total of 137 differentially expressed proteins were identified under SM stress, of which 70 were up-regulated and 67 were down-regulated. Bioinformatics analysis indicated that several metal pathways, such as metal cluster binding, iron-sulfur cluster binding, and transition metal ion binding, may affect the resistance of A. hydrophila to SM. Finally, the sensitivity of 6 related gene deletion strains to streptomycin was evaluated. Overall, the results of this study provide a deeper understanding of the response behavior of A. hydrophila to streptomycin stress.