Nocardia seriolae is one of the most important bacterial pathogens in cultured fish, causing serious necrotic granulomas in tissues. As a Gram-positive bacterium belonging to the order Actinomycetales, the pathogenic mechanism of N. seriolae is complex and not well defined. Two-component systems (TCS) function as signal transducers in prokaryotes that respond to different environmental stimuli. In this study, a novel two-component system gene, mprA/B homolog (mprA/B1), was identified in N. seriolae and a double gene-deficient mutant was generated. The ΔmprA/B1 strain exhibited faster growth and a more turbid bacterial suspension in trypticase soy broth (TSB). Transmission electron microscopy revealed an extra irregular polysaccharide layer outside the cell wall in the ΔmprA/B1 strain. Moreover, the ΔmprA/B1 strain showed a significantly increased sensitivity to macrolides and rifampicin. In a cell line, both strains were able to invade and survive intracellularly and induce cell apoptosis, but the ΔmprA/B1 strain showed lower cell invasion and intracellular survival rates. In largemouth bass, the ΔmprA/B1 strain showed significantly less tissue bacterial load and attenuated virulence. RNA sequencing identified 492 upregulated and 851 downregulated genes in the ΔmprA/B1 strain, and the gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment showed that many upregulated genes were involved in sugar and amino acid metabolic processes, and many downregulated genes were involved in drug efflux pump and virulence factor. These findings provide functional insights into the role of MprA/B in N. seriolae.