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<i>Pseudomonas plecoglossicida</i> is the pathogen of visceral white spot disease in marine fish, which usually occurs at 16–19 °C and has resulted in heavy economic losses. Our previous RNA sequencing revealed that the expression of the <i>fliL</i> gene in <i>P. plecoglossicida</i> was significantly up-regulated during infection of the host. In order to study the influence of the <i>fliL</i> gene on the virulence of <i>P. plecoglossicida</i>, the <i>fliL</i> gene of the NZBD9 strain was knocked out by the homologous recombination method, the <i>fliL</i> gene-deleted strain (Δ<i>fliL</i> strain) constructed, and complemented the <i>fliL</i> gene to the Δ<i>fliL</i> strain to obtain the C-Δ<i>fliL</i> strain. The growth curves of the NZBD9 strain, Δ<i>fliL</i> strain, and C-Δ<i>fliL</i> strain did not show significant differences. Compared with the NZBD9 strain, the motility, adhesion, and biofilm formation ability were tendered in the Δ<i>fliL</i> strain (<i>p</i> < 0.05); the complement of the <i>fliL</i> gene enhanced these abilities to the level of the NZBD9 strain. The results of artificial infection experiments showed that the LD₅₀ of NZBD9 strain, Δ<i>fliL</i> strain, and C-Δ<i>fliL</i> strain in hybrid grouper (<i>Epinephelus fuscoguttatus</i> ♀ × <i>E. lanceolatus</i> ♂) were 5.0 × 10³ CFU/fish, 6.3 × 10⁴ CFU/fish, and 1.3 × 10³ CFU/fish, respectively. RNA sequencing was performed on wild-type strains and Δ<i>fliL</i> strains. A total of 126 differentially expressed genes (DEGs) were screened (<i>p</i> < 0.05), of which 114 were downregulated and 12 were upcontrolled, among which several genes related to the six-type secretion system and transport activity were significantly downregulated. The DEGs were aligned to the GO and KEGG databases and enriched to 44 GO pathways and 39 KEGG pathways, respectively. The active pathways of ABC transporters were significantly enriched in both databases. These results indicate that the <i>fliL</i> gene is related to the movement, biofilm formation, and adhesion ability of <i>P. plecoglossicida</i>, and may reduce virulence by affecting substance transport and bacterial secretion.