Glaesserella parasuis is an important bacterial pathogen that affects the swine industry worldwide. Research on the pathogenic mechanism and genetically engineered vaccine remains undeveloped because an effective markerless and multiple-gene knockout system is unavailable for G. parasuis yet. To establish a markerless knockout, deleted allelic genes with kanamycin resistance (Kan R ) cassettes were introduced into the genome of G. parasuis by using natural transformation with suicide plasmids. Then, the Kan R cassette was excised with a thermosensitive plasmid pGF conferring a constitutive Flp expression. To realize the markerless and multiple-gene knockout, plasmid pGAF was constructed by placing the Flp gene under the control of an arabinose-inducible promoter. Firstly, pGAF was introduced into G. parasuis by electroporation, and the marked mutants were produced following natural transformation. Finally, the Kan R cassette was excised from the genome by the inducible expression of Flp upon arabinose action. Based on the natural transformation and the inducible expression of Flp, the markerless single-gene knockout mutants of Δ hsdR , Δ neuA2 , Δ espP2 , Δ apd , and Δ nanH were constructed. In addition, a five-gene knockout mutant of Δ hsdR Δ neuA2 Δ espP2 Δ apd Δ nanH was generated by successive natural transformation with five suicide plasmids. Taken together, a markerless and multiple-gene deletion system was established for G. parasuis in the present study for the first time. This system is simple, efficient, and easy to manipulate for G. parasuis ; thus, our technique will substantially aid the understanding of the etiology, pathogenesis, and genetic engineering of G. parasuis and other bacteria that can be naturally transformed in laboratory conditions. Key points • Flp recombinase excised the Kan R  gene flanked by FRT sites in  Glaesserella parasuis. • The regulatory expression of Flp enabled a multiple-gene knockout for G. parasuis. • The technique will promote the understanding of Glässer’s disease pathogens. Graphical abstract