Pseudomonas plecoglossicida is the pathogen of visceral white spot disease in large yellow croaker (Larimichthys crocea). At present, the pathogenic mechanism of P. plecoglossicida has been partially understood, and some effective vaccines have been screened, whereas no commercial vaccine has been developed. [Objective] To deepen the understanding about the pathogenic mechanism and develop efficient attenuated live vaccines of P. plecoglossicida. [Methods] We targeted luxR and the RNA polymerase σ factor gene rpoE associated with quorum sensing to construct deletion mutants of P. plecoglossicida NB2011 by double homologous recombination. In addition, the strain ΔT6SS1ΔluxR with deletion of both the type VI secretion system 1 (T6SS1) gene and luxR was constructed. The biological characteristics and virulence of the mutants were analyzed. The relative percent of survival of fish after vaccination with the double deletion mutant was investigated. [Results] We successfully constructedΔluxR, ΔT6SS1ΔluxR, and ΔrpoE. Compared with the wild type, none of the three mutants showed significant changes in the growth rate, swarming ability or swimming ability, while ΔluxR and ΔT6SS1ΔluxR showed significant decreases in biofilm formation. The internalization, adsorption, and intracellular proliferation of the mutants in mouse macrophages were observed. All the three mutants could survive in J774A.1 macrophages and showed lower proliferation capacities than the wild type. Compared with the wild type, the two single mutants showed significantly reduced virulence to goldfish (Carassius auratus) after intraperitoneal injection at a concentration of 1.0×10⁷ cells/mL. The double deletion mutant ΔT6SS1ΔluxR showed no virulence when challenging goldfish at the same dose, with the LD₅₀>10⁸ cells/mL. In the immune protection experiment, the goldfish was vaccinated with ΔT6SS1ΔluxR, and the fish was artificially challenged with the wild type 28 days later. The relative percent of survival reached 78.60%, which indicated effective protection. [Conclusion] In this study, three gene deletion mutants were successfully constructed, and the virulence of the mutants was significantly decreased. Among them, ΔT6SS1ΔluxR is expected to be a candidate strain for the development of the attenuated live vaccine against P. plecoglossicida infection.