〔Abstract〕 Objective To use linear PCR fragment containing antibiotic resistance cassette to carry out homologous recombination and replacement of target gene fragment ofAcinetobacter baumanniito achieve rapid gene knockout and functional verification. Methods Acinetobacter baumanniiAb4294 was used as the research object, and the upper(901 bp) and lower(1 028 bp) reaches offimgene cluster(4 980 bp in length) were amplified by PCR, which was used as the recombinant homologous arm. Kanamycin antibiotic resistance cassette(KanR) was amplified from pUC57 plasmid. The above three fragments were connected by overlapping extended PCR technique, and the connected fragments were transformed into wildAcinetobacter baumanniistrains. The gene deletion mutant was screened, and the plasmid complement strain was constructed. The phenotype of the obtained strains was identified, and the function offimgene cluster was explored. Results A mutant strain ofAcinetobacter baumanniiAb4294 with deletion offimgene cluster was successfully constructed by homologous substitution of linear PCR fragment containing antibiotic resistance cassette. Compared with the wild strain, the growth curve of the deletion strain had no significant difference, and the rubbing ability significantly decreased, and the phenotype recovered after complementing the gene cluster. Conclusion Thefimfamily genes ofAcinetobacter baumanniiAb4294 is successfully knocked out by homologous substitution of linear PCR fragment containing antibiotic resistance cassette, which encodes the product involved in the motile movement ofAcinetobacter baumannii.