A number of different strategies have been developed to achieve Site-directed mutagenesis. One of these methods is in-frame deletion technique which efficiently creates mutations at specific sites by deletion a number of nucleotides from a bacterial chromosome. The fhuCD operon consists of two genes (fhuC and fhuD) which are involved in the uptake of ferric hydroxamate siderophores across the bacterial membranes. In the present study, a construction of the EiΔfhuCD mutants was conducted. EiΔfhuCD mutant was achieved by performing the in-frame fhuCD fragment through overlapping extension PCR then digested the in-frame fragment by SacI and XbaI restriction enzyme and ligated to the suicide plasmid pMEG-375. The recombination plasmid (pEiΔfhuCD) was transformed into E. ictaluri wild type strain by conjugation and integrated into the genomic DNA through two steps of homologous recombination. The colony PCR and DNA sequencing were used to genotypically confirm the deletion. Besides, it was successfully deleted a 1635bp/545aa from a 1707-bp of the fhuCD operon.