Update publication information. Aeromonas hydrophila is an aquatic pathogen of freshwater fish. The emergence of widespread antimicrobial-resistance strains of this pathogen has caused increasing rates of fish infections. Our previous research reported that A. hydrophila yeeY, a LysR-type transcriptional regulator (LTTR), negatively regulated furazolidone (FZ) resistance, although its intrinsic regulatory mechanism is still unclear. In this study, a DIA-quantitative proteomics method was used to compare the differentially expressed proteins (DEPs) between ΔyeeY and the wild‐type strain under furazolidone treatment. When compared to the control, a total of 594 DEPs were identified in ΔyeeY, of which 293 proteins were increased and 301 were decreased in abundance. Bioinformatics analysis showed that several biological pathways, such as the secretion system and protein transport, were involved in FZ resistance. Subsequent antibiotics susceptibility assays of several gene deletion strains identified from the proteomics results showed that YeeY may regulate cysD, AHA_2766, AHA_3195 and AHA_4275 to affect the FZ resistance. Moreover, 34 antimicrobial resistance genes (ARGs) from the bacterial drug resistance gene database (CARD) were found to be directly or indirectly regulated by YeeY as well. A subsequent assay of several ARGs mutants showed that ΔAHA_3222 increased while ΔcysN and ΔAHA_3753 decreased the susceptibility of A. hydrophila to FZ. Finally, AHA_3222 and AHA_4275 were found to be directly transcriptionally regulated by YeeY from chromatin immunoprecipitation (ChIP) PCR and an electrophoretic mobility shift assay (EMSA). Taken together, our findings demonstrated that YeeY participates in antimicrobial resistance of A. hydrophila to furazolidone, and the results provide new targets for the development of novel antimicrobial agents in the future.