In bacteria fatty acids are synthesized by type II fatty acid synthase system, in which 3-hydroxylacyl -ACP dehydratase is one of the key enzymes for bacterial growth. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease to all cruciferous plants, and great economic losses to the world. To study the 3-hydroxylacyl-ACP dehydratase in Xcc, XC_2876 (XcfabZ) was found based on the sequence alignment with E. coli FabZ. Moreover, XcFabZ shows 46.1% amino acid sequence identity with EcFabZ, and contains the conservative α-helix structure and active residue. XcfabZ was able to genetically complement the EcfabZ knock out mutant E.coli HW7 to restore the growth with inducer IPTG. In vitro assay also identified XcFabZ was able to dehydrated 3-hydroxyacyl-ACP in the initial and elongation reactions. However, XcfabZ in the chromosome could not be deleted directly, indicating XcfabZ is essential for growth. When expression plasmids harboring EcfabZ or XcfabZ was introduced, respectively, XcfabZ deletion mutants were constructed. The EcfabZ replaced mutant showed different fatty acid compositions, much lower tolerance to stressful conditions (high salty, low pH, H₂O₂ and SDS), and decreased motility compared to the wild-type. While mutant with XcfabZ in the plasmid showed similar phenotype to wild-type. These data demonstrated both XcFabZ and EcFabZ show 3-hydroxylacyl-ACP dehydratase activity, but they may contains different biological function in vivo.