[Objective] Microbial activities are considered the main cause of most food spoilage. Study of the regulation mechanism of spoilage is valuable to maintain quality and safety of food. Pseudomonas fluorescens is a typical Gram-negative spoilage bacterium. The aim of this study was to elucidate the role of the alternative sigma factor RpoS in spoilage caused by P. fluorescens.[Methods] An rpoS mutant of P. fluorescens isolated from refrigerated fish was constructed by homologous recombination. Survival rates of the wild type and the mutant under different stress conditions were compared. The types and contents of extracellular autoinducers acylated homoserine lactones (AHLs) were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, the wild type and the mutant were inoculated into the sterile salmon juice stored at 4℃, and total viable count (TVC) and total volatile basic nitrogen (TVB-N) were assayed.[Results] The in-frame deletion rpoS mutant strain was successfully constructed in P. fluorescens. Deletion of rpoS gene significantly decreased the resistance to 10 mmol/L H₂O₂and 15% alcohol, increased the resistance to 150 μg/mL crystal violet and 175 mmol/L acetic acid, and had no influence on the resistance to 47℃ and 20% NaCl. The ropS mutant produced higher contents of long chain AHLs C₁₀-HSL, C₁₂-HSL and C₁₄-HSL than the wild type one. In addition, the mutant reduced TVB-N production in sterilized salmon juice stored at 4℃.[Conclusion] These results indicated that RpoS was not only involved in the resistance to multiple stresses, but also influenced quorum sensing and spoilage potential of P. fluorescens.