Abstract: Burkholderia pyrrocinia JK-SH007 is an effective biocontrol agent against poplar canker. Based on the genomic data of JK-SH007 strain, specific primers were designed to clone the ssuA gene, and bioinformatics were used to analyze the similarity, phylogeny, and physicochemical properties of the ssuA protein to homologous proteins of other species, and the ssuA knockout mutant of JK-SH007(JK-SH007∆ssuA) was obtained by double crossover of homologous recombination. On this basis, the growth curve, motility, exopolysaccharide, siderophore production, cyclic Dimeric Guanosine Monophosphate (c-di-GMP), biofilm formation, antibacterial ability, expression of prn gene cluster and endophytic colonization dynamics of wild-type strain (WT) and knockout strain were determined. The results showed that the full-length sequence of ssuA gene is 1107 bp, encoding 366 amino acids, and the secondary structure and tertiary structure of the encoded protein are identical. Phylogenetic analysis showed that the ssuA protein was the most closely related to Burkholderia arboris. The exopolysaccharide synthesis, biofilm formation, siderophore production and antibacterial ability of the ∆ssuA strain were weaker than those of WT, but the growth ability was not changed. Two genes (prnA and prnB) were significantly down-regulated, while prnC and prnD were up-regulated. The content of c-di-GMP decreased by 4.97%. The endophtic colonization ability of roots, stems and leaves of poplar tissue-cultured seedlings decreased by 78.57%, 80.77% and 66.67%, respectively. Therefore, the ssuA gene positively regulated biofilm formation of JK-SH007, which was closely related to exopolysaccharide production, siderophore production and c-di-GMP synthesis, and affected the antibacterial ability and endogenous colonization ability, but had no effect on the growth of the strain. This study lays a foundation for revealing the effect of ssuA gene on the colonization and biocontrol ability of strain JK-SH007, and also provides a scientific basis for the development and efficient application of biocontrol microbial agents for poplar canker disease.