Long-chain unsaturated fatty acids (UFAs) can serve as nutrient sources or building blocks for bacterial membranes. However, little is known about how UFAs may be incorporated into the virulence programs of pathogens. A previous investigation identified FabR as a positive regulator of virulence gene expression in Edwardsiella piscicida. Here, chromatin immunoprecipitation-sequencing coupled with RNA-seq analyses revealed that 10 genes were under the direct control of FabR, including fabA, fabB, and cfa, which modulate the composition of UFAs. The binding of FabR to its target DNA was facilitated by oleoyl-CoA and inhibited by stearoyl-CoA. In addition, analyses of enzyme mobility shift assay and DNase I footprinting with wild-type and a null mutant (F131A) of FabR demonstrated crucial roles of FabR in binding to the promoters of fabA, fabB, and cfa. Moreover, FabR also binds to the promoter region of the virulence regulator esrB for its activation, facilitating the expression of the type III secretion system (T3SS) in response to UFAs. Furthermore, FabR coordinated with RpoS to modulate the expression of T3SS. Collectively, our results elucidate the molecular machinery of FabR regulating bacterial fatty acid composition and virulence in enteric pathogens, further expanding our knowledge of its crucial role in host-pathogen interactions.