The RNA chaperone Hfq plays a pivotal role in various bacterial physiological functions. In Aeromonas hydrophila, a prevalent fish pathogen, Hfq was found to undergo acyl modification, yet the precise roles of Hfq and modifications at the K56 site remained unknown. Physiological assays revealed that the hfq deletion and succinylation modification on K56 residue of Hfq protein affected various phenotypes including biofilm formation, protease activity, chemotaxis and stress resistance.. Label-free quantitative proteomics technology was used to analyze protein intensity changes in hfq derivatives. The resulting DEPs showed that the Hfq group (Δhfq vs. WT) had 1053 DEPs (396 up, 657 down), and the K56E group (K56E vs. K56R) had 418 DEPs (212 up, 206 down). GO and KEGG analyses of these DEPs revealed that Hfq deletion altered proteins related to oxidative phosphorylation, peptide transport, chemotaxis, and fatty acid degradation, while Hfq K56E modification affected proteins involved in anaerobic respiration, oxidative phosphorylation, and pyruvate metabolism. This study confirms the multifaceted functions of hfq and elucidates the complex regulatory mechanisms of K56 succinylation in various physiological functions of A. hydrophila.