Simple Summary: Aeromonas hydrophila is a major pathogen with human–animal–fish comorbidity that causes many diseases. To better investigate the roles of different genes in Aeromonas hydrophila, our aim is to knock out a gene in A. hydrophila and study whether the gene affects the physiological phenotypes or not. We found that AHA_2124, encoding a multiple antibiotic resistance regulator (MarR) family regulator, affected the extracellular protease activity, hemolytic activity, and motility of the phytopathogenic bacterium Aeromonas hydrophila. The MarR family of the transcriptional regulators of bacteria are mainly involved in the regulation of various cellular processes. In this work, we call for further research on the bioinformatics analysis based on the comparison of the ΔAHA_2124 strain and wild type. The transcriptional regulators of the MarR family play an important role in diverse bacterial physiologic functions, whereas their effect and intrinsic regulatory mechanism on the aquatic pathogenic bacterium Aeromonas hydrophila are, clearly, still unknown. In this study, we firstly constructed a deletion strain of AHA_2124 (ΔAHA_2124) of a MarR family transcriptional regulator in Aeromonas hydrophila ATCC 7966 (wild type), and found that the deletion of AHA_2124 caused significantly enhanced hemolytic activity, extracellular protease activity, and motility when compared with the wild type. The differentially abundant proteins (DAPs) were compared by using data-independent acquisition (DIA), based on a quantitative proteomics technology, between the ΔAHA_2124 strain and wild type, and there were 178 DAPs including 80 proteins up-regulated and 98 proteins down-regulated. The bioinformatics analysis showed that the deletion of gene AHA_2124 led to some changes in the abundance of proteins related to multiple biological processes, such as translation, peptide transport, and oxidation and reduction. These results provided a theoretical basis for better exploring the regulatory mechanism of the MarR family transcriptional regulators of Aeromonas hydrophila on bacterial physiological functions. [ABSTRACT FROM AUTHOR]