Post-translational modifications are crucial in regulating biological functions across both prokaryotes and eukaryotes. In Aeromonas hydrophila, CobQ, a recently identified novel deacetylase, plays a significant role in lysine deacetylation, influencing bacterial metabolism and stress responses. The present study utilized quantitative proteomics to investigate the impact of cobQ deletion on the global protein expression profile in A. hydrophila. Through dataindependent acquisition mass spectrometry, we identified 233 upregulated and 41 downregulated proteins in the cobQ deletion mutant (ΔahcobQ) strain compared to the wild-type (WT) strain. Key differentially expressed proteins were involved in oxidative phosphorylation, bacterial secretion, and ribosomal function. Additionally, phenotypic assays demonstrated that the ΔahcobQ strain exhibited an increased resistance to oxidative phosphorylation inhibitors, suggesting a pivotal role for AhCobQ in energy metabolism. Outer membrane proteins and efflux pumps also showed altered expression, indicating potential implications for membrane permeability and antibiotic resistance. These results suggested that AhCobQ plays a vital regulatory role in maintaining metabolic homeostasis and responding to environmental stress, highlighting its potential as a target for therapeutic interventions against A. hydrophila infections.