Edwardsiella piscicida is a gram-negative bacterial pathogen invading a wide range of fish species. Response regulator EsrB is essential for the activation of type III and type VI secretion systems (T3/T6SS). In this study, proteomes of the wild-type E. piscicida EIB202 and ΔesrB mutant strains were compared to identify the regulon components of EsrB cultured in DMEM allowing T3/T6SS expression. As a result, 19 proteins showed different expression, which were identified to be associated with T3/T6SS, related to amino acid transport and metabolism, and energy production. Particularly, GlnA, a glutamine synthetase essential for ammonia assimilation and glutamine biosynthesis from glutamate, was found to be regulated negatively by EsrB. Moreover, GlnA affected bacterial growth in vitro and bacterial colonization in vivo. Collectively, our results indicated that EsrB plays important roles in regulating the expression of metabolic pathways and virulence genes, including glutamine biosynthesis in E. piscicida during infection.