Pseudomonas plecoglossicida, the causative agent of white spot disease of large yellow croaker, has caused serious economic losses to the aquaculture industry. The type VI secretion system (T6SS) is a significant virulence system widely distributed among Gram‐negative bacteria. VgrG, a structural and core component of T6SS, is crucial to the function of T6SS. To explore the biological profiles mediated by vgrG gene and its effects on the pathogenicity of P. plecoglossicida, the vgrG gene deletion (ΔvgrG) strain and complementary (C‐ΔvgrG) strain were constructed and the differences in pathogenicity and virulence‐related characteristics between different strains were analysed. The results showed that vgrG gene deletion significantly affected the virulence‐related characteristics of P. plecoglossicida, including chemotaxis, adhesion, and biofilm formation. In addition, the LD50 of ΔvgrG strain was nearly 50‐fold higher than that of the NZBD9 strain. Transcriptome data analysis suggested that the vgrG gene may affect the virulence of P. plecoglossicida by regulating the quorum sensing pathway to inhibit the secretion of virulence factors and affect biofilm formation. Besides, deletion of the vgrG gene may reduce bacterial pathogenicity by affecting bacterial signal transduction processes and the ability to adapt to chemotactic substances.