Abstract: The pollution of aquaculture water by spoilage bacteria can easily lead to a deterioration in the quality of aquatic products. In this study, Bacillus subtilis C15, isolated from tilapia, and its heat-inactivated postbiotics were used to investigate their effects on improving the quality of tilapia meat in aquaculture, as well as their potential mechanisms. Both C15 and its postbiotics were found to effectively inhibit the adhesion ability of tilapia-derived Pseudomonas putida (LP-3) to the intestinal mucus. To further assess their effects on the meat quality of tilapia aquacultured in LP-3 polluted water, tilapias were randomly divided into four groups: normal (Con), model (LP-3), live bacteria (HC15), and postbiotic (SC15). The spoilage model was induced by adding Pseudomonas putida LP-3 to the water to reach a concentration of 10⁴ CFU/mL, while 10⁴ CFU/mL of Bacillus subtilis C15 (HC15) or an equivalent dose of postbiotics (SC15) were added to the water for intervention. The experimental period was 20 days. The results showed that LP-3 pollution significantly reduced meat quality, while the addition of C15 and postbiotics improved the meat quality to varying degrees. The hardness of the fish meat increased by 63.7% and 48.7%, respectively, and the chewiness nearly doubled. After five days of iced storage, the TVB-N content was reduced to below 30 mg/100 g. Moreover, compared to the live bacteria group, the postbiotic group showed higher SOD enzyme activity and a greater concentration of high-threshold alkanes (such as tetradecane, pentadecane, and nonadecane) in the volatile flavor compounds of tilapia meat. Analysis of the gut microbiota revealed that the intervention with C15 and its postbiotics modulated the structure and composition of the gut microbiota in tilapia, significantly reducing the relative abundance of strains highly associated with the production of off-flavors, such as Pseudomonas and Agromyces (P<0.05). Additionally, the gut microbiota was found closely associated with flavor. The above results indicated that C15 and its postbiotics could inhibit the adhesion of Pseudomonas putida in tilapia and improve the quality of tilapia under spoilage condition. The potential mechanism might be related to enhancing the antioxidant capacity of the meat and maintaining the balance of the gut microbiota. This study provides a reference for the research and application of Bacillus subtilis and its postbiotics in aquaculture health management and quality regulation.