ABSTRACTAvibacterium paragallinarumis the pathogen involved in infectious coryza (IC), an acute infectious upper respiratory disease in chickens. The prevalence of IC has increased in China in recent years. There is a lack of reliable and effective procedures for gene manipulation, which has limited the research on the bacterial genetics and pathogenesis ofA. paragallinarum. Natural transformation has been developed as a method of gene manipulation inPasteurellaceaeby the introduction of foreign genes or DNA fragments into bacterial cells, but there has been no report on natural transformation inA. paragallinarum. In this study, we analyzed the existence of homologous genetic factors and competence proteins underlying natural transformation inA. paragallinarumand established a method for transformation in it. Through bioinformatics analysis, we identified 16 homologs ofHaemophilus influenzaecompetence proteins inA. paragallinarum. We found that the uptake signal sequence (USS) was overrepresented in the genome ofA. paragallinarum(1,537 to 1,641 copies of the core sequenceACCGCACTT). We then constructed a plasmid, pEA-KU, that carries the USS and a plasmid, pEA-K, without the USS. These plasmids can be transferred via natural transformation into naturally competent strains ofA. paragallinarum. Significantly, the plasmid that carries USS showed a higher transformation efficiency. In summary, our results demonstrate thatA. paragallinarumhas the ability to undergo natural transformation. These findings should prove to be a valuable tool for gene manipulation inA. paragallinarum.IMPORTANCENatural transformation is an important mechanism for bacteria to acquire exogenous DNA molecules during the process of evolution. Additionally, it can also be used as a method to introduce foreign genes into bacteria under laboratory conditions. Natural transformation does not require equipment such as an electroporation apparatus. It is easy to perform and is similar to gene transfer under natural conditions. However, there have been no reports on natural transformation inAvibacterium paragallinarum. In this study, we analyzed the presence of homologous genetic factors and competence proteins underlying natural transformation inA. paragallinarum. Our results indicate that natural competence could be induced inA. paragallinarumserovars A, B, and C. Furthermore, the method that we established to transform plasmids into naturally competentA. paragallinarumstrains was stable and efficient.