Yersinia ruckeri is a conditional pathogenic bacterium which has widespread pathogenicity. The most important virulence regulatory system of Y. ruckeri is its type III secretion system (T3SS), while invF gene is an important regulatory factor of the T3SS. In this study, we constructed an unmarked deletion mutant with invF gene missing of a Y. ruckeri SC09, a highly virulent strain isolated from Ictalurus punctatus, and studied its biological characteristics. In order to investigate the effects of invF and T3SS on the pathogenicity of Y. ruckeri, construction of homologous arm AC, upstream fragment A and downstream fragment C of invF gene, was combined in overlap extension PCR. Then the homologous arm AC was ligated with the pLP12 suicide vector to generate the plasmid pLP12-invF. The plasmid was transformed into E. coli DH5α λ pir cells and it was amplified. The pLP12-invF was extracted and transformed into E. coli β2163 by electroporation, designated pLP12-invF-β2163. Then the pLP12-invF was transferred into Y. ruckeri SC09 strain through conjugation. Using the chloramphenicol for screening of insertional mutants and the vmt gene with L-arabinose for counter selection of deletion mutants. The deletion mutants were confirmed by PCR and subsequent sequencing. Then we observed morphology of bacteria and colonies, identified biochemical characterization and measured growth curve of the deletion mutants and wild type.In this study, the Y. ruckeri SC09 strain invF gene has been successfully knockouted. The morphology of bacteria and colonies, biochemical characteristics of deletion mutants and wild type were similar, but the colony size of deletion mutants was smaller than wild type and the growth of deletion mutants was slower than that of wild type. The method of pLP12 suicide vector, a highly efficient conjugation system form E. coli β2163 and screening by the antibiotic coupled with counter selecting by vmt gene, can be a simple and efficient gene editing operation to deal with Y. ruckeri, in the absence of a significant effect on its basic biological characteristics. In conclusion, this research obtained the unmarked mutant genes with the missing invF successfully, which laid a foundation for the studies of the pathogenicity of Y. Ruckeri in the future.