BACKGROUND: The contributive role of the microbiome in tumor progression has been reported in multiple studies, such as the Fusobacterium nucleatum (F. nucleatum) in breast cancer (BC). This study aimed to explore the role of F. nucleatum-derived small extracellular vesicles (Fn-EVs) in BC and preliminarily uncover the mechanism. METHODS: Ten normal and 20 cancerous breast tissues were harvested to investigate the gDNA expression of F. nucleatum and its relation with the clinical characteristics of BC patients. After isolating Fn-EVs by ultracentrifugation from F. nucleatum (ATCC 25,586), both MDA-MB-231 and MCF-7 cells were treated with PBS, Fn, or Fn-EVs, followed by being subjected to CCK-8, Edu staining, wound healing, and Transwell assays to detect their cell viability, proliferation, migration, and invasion. TLR4 expression in BC cells with diverse treatments was assessed by western blot. In vivo experiments were performed to verify its role in tumor growth and liver metastasis. RESULTS: The F. nucleatum gDNA levels of breast tissues in BC patients were significantly higher than those in normal subjects, and positively associated with tumor size and metastasis. Fn-EVs administration significantly enhanced the cell viability, proliferation, migration, and invasion of BC cells, while knocking down TLR4 in BC cells could block these effects. Furthermore, in vivo study verified the contributive role of Fn-EVs in tumor growth and metastasis of BC, which might rely on its regulation of TLR4. CONCLUSIONS: Collectively, our results suggest that F. nucleatum plays an important role in BC tumor growth and metastasis by regulating TLR4 through Fn-EVs. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.