Methods including insertion mutagenesis with transposon mTn5gusA-pgfp21-, inverse PCR, DNA sequencing, Southern blot, motility tests and Biolog ECO were used to study the motility correlated genes in Klebsiella oxytoca KO108. The results showed that a mutant library of KO108 with Kan resistance, GUS and GFP marker was constructed by conjugating KO108 with pFAJ1819∶∶21/Escherichia coli S17.1. Two strains with striking decrease in motility ability, named as MA and MD, were screened from 100 mutants randomly selected from the library. The solo insertion of transposon mTn5gusApgfp21 in the MA and MD was verified with Southern blot by using gfp as probe. The size of fragments inserted by mTn5gusApgfp21 was confirmed with the same method. Results of motility tests showed that the movement ability of KO108 was distinctly stronger than that of MA and MD(P<0.05). There was no significant difference of movement ability between MA and MD. Results of inverse PCR and DNA sequencing proved that the flanking sequence of mTn5gusA pgfp21 integration sites in the genome of MA was homologous with gene of NAD(P)H quinone oxidoreductase(NqrA) for the MA and the flanking sequence of mTn5gusApgfp21 integration sites in the genome of MD was homologous with the gene cluster of lipopolysaccharide synthesis for the MD. The growth curves among the strains of MA, MD and KO108 were not obviously different. Results of velum tests showed that MD was different from KO108 and MA, but it could not be distinguished easily between the later two. Results of assaying carbon source utilization with Biolog ECO showed that MA and MD could not use gamma D galactose acid lactone and 2 hydroxy benzoic acid in 72 h. It will lay a solid foundation for further studying genes involved in motility of K. oxytoca.