Walnut bacterial blight caused by Xanthomonas arboricola pv. juglandis ( Xaj ) is one of the most prevalent diseases of walnut ( Juglans spp.), causing significant reductions in nut yield and important losses in economy. Enoyl-acyl carrier protein (ACP) reductase (ENR) is one of the key enzymes involved in the biosynthesis of bacterial fatty acids. In this study, we identified a single ENR-encoding gene, RS10040, in the genome of the Xaj DW3F3 strain. Sequence alignment analysis suggested RS10040 as a candidate fabV gene in Xaj . Expression of XajfabV restored the growth of the Escherichia coli fabI temperature-sensitive mutant under a nonpermissive growth condition. In vitro assays demonstrated that Xaj FabV catalyzed enoyl-ACPs of various chain lengths to acyl-ACPs, demonstrating its role in de novo fatty acid biosynthesis. Furthermore, we confirmed that XajfabV is an essential gene for growth, as no XajfabV deletion mutant could be obtained, although XajfabV in the chromosome could be deleted after compensating with a functional ENR-encoding gene via an exogenous plasmid. The fabV replacement mutants showed similar growth characteristic and fatty acid compositions. Our data further identified that fabV conferred Xaj with tolerance to various environmental stresses. Although Xaj FabV conferred Xaj with triclosan resistance, the resistance of Xaj was weaker than that found for Pseudomonas aeruginosa . Moreover, triclosan exhibited a control effect against infection of the Δ fabV/EcfabI to its host walnut. This study revealed the function of Xaj FabV and laid a theoretical foundation for the fatty acid synthesis mechanism of Xaj ., Competing Interests: The author(s) declare no conflict of interest.