Abstract: Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker (Larimichthys crocea) and orange-spotted grouper (Epinephelus coioides). Previously, RNA sequencing showed that P. plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection. To explore the role of flgK in P. plecoglossicida pathogenicity, RNA interference (RNAi) was performed to silence the P. plecoglossicida flgK gene, and the mutant (flgK-RNAi strain) with the best silencing efficiency (89.40%) was chosen for further study. Results showed that flgK gene silencing significantly attenuated P. plecoglossicida motility, adhesion, and biofilm formation. Compared to those fish infected with the wild-type strain of P. plecoglossicida, orange-spotted grouper infected with the flgK-RNAi strain showed a 55% increase in the survival rate and a one-day delay in time of first death, with fewer pathogens in the spleen and fewer white spots on the spleen surface. RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase (MAPK) signaling pathway was related to multiple immune-related pathways. Furthermore, arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways. These findings suggest that flgK is a virulence gene of P. plecoglossicida. Furthermore, flgK appears to be involved in the regulation of motility, adhesion, and biofilm formation in P. plecoglossicida, as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P. plecoglossicida infection.