Enoyl-ACP reductase (ENR) is one of the key enzymes in bacterial fatty acids biosynthesis. Through sequence alignment, gene XC_0119, annotated as trans-2-enoyl CoA reductase, was found in the genome of Xanthomonas campestris pv. campestris (Xcc) 8004. However, the protein encoded by XC_0119 shows 59% identity with fabV, the ENR from Pseudomonas aeruginosa, and contains the same catalytic triad Tyr-(Xaa)₈-Lys. Expression of XccfabV restored the growth of the E. coli fabI temperature sensitive mutant JP1111 under non-permissive condition. In vitro assay identified that XccFabV catalysed enoyl-ACP with viarable chain lengths to acy-ACP, and the activity was not inhibited by triclosan. Furthermore, genetic research proved that XccfabV is an essential gene, and none of XccfabV deletion mutants was obtained. However, XccfabV in the chromosome could be deleted when plasmid expressing E. coli fabI was introduced into Xcc8004. And the fabI replaced mutant showed similar growth characteristic and fatty acid compositions with wild type, but changed to be sensitive to triclosan. These results domonstrated XccfabV is essential for growth, encodes ENR involved in fatty acid de novo biosynthesis, and FabV confers triclosan resistance on Xcc.