Currently, the influence of the tumor microbiome on the effectiveness of immunotherapy remains largely unknown. Intratumoural Fusobacterium nucleatum (Fn) functions as an oncogenic bacterium and can promote tumor progression in esophageal squamous cell carcinoma (ESCC). Our previous study revealed that Fn is a facultative intracellular bacterium and that its virulence factor Fn-Dps facilitates the intracellular survival of Fn. In this study, we find that Fn DNA is enriched in the nonresponder (NR) group among ESCC patients receiving PD-1 inhibitor and that the serum antibody level of Fn is significantly higher in the NR group than in the responder (R) group. In addition, Fn infection has an opposite impact on the efficacy of αPD-L1 treatment in animals. Mechanistically, we confirm that Fn can inhibit the proliferation and cytokine secretion of T cells and that Fn-Dps binds to the PD-L1 gene promoter activating transcription factor-3 (ATF3) to transcriptionally upregulate PD-L1 expression. Our results suggest that it may be an important therapeutic strategy to eradicate intratumoral Fn infection before initiating ESCC immunotherapies.