AbstractHuanglongbing (HLB) is a destructive citrus disease. The leading cause of HLB is Candidatus Liberibacter asiaticus. Fatty acid biosynthesis is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterial agents. Enoyl−acyl carrier protein reductase (also called ENR or FabI and a product of the fabI gene) is an enzyme required in a critical step of bacterial fatty acid biosynthesis and has attracted attention as a target of novel antimicrobial agents. We determined the crystal structures of FabI from Ca. L. asiaticus in its apoform as well as in complex with b‐nicotinamide adenine dinucleotide (NAD) at 1.7 and 2.7 Å resolution, respectively, to facilitate the design and screening of small molecule inhibitors of FabI. The monomeric ClFabI is highly similar to other known FabI structures as expected; however, unlike the typical tetramer, ClFabI exists as a hexamer in crystal, whereas as dimer in solution, on the other hand, the substrate binding loop which always disordered in apoform FabI structures is ordered in apo‐ClFabI. Interestingly, the structure of ClFabI undergoes remarkable conformational change in the substrate‐binding loop in the presence of NAD. We conclude that the signature sequence motif of FabI can be considered as Gly‐(Xaa)5‐Ser‐(Xaa)n‐Val‐Tyr‐(Xaa)6‐Lys‐(Xaa)n‐Thr instead of Tyr‐(Xaa)6‐Lys. We have further identified isoniazid as a competitive inhibitor with NADH.