Fusobacterium nucleatum is an oral pathogen associated with periodontal disease, preterm births and the exacerbation of colorectal, esophageal, pancreatic, and breast cancers. F. nucleatum was previously found to inhibit killing of cancer cells by natural killer (NK) cells and tumor infiltrating T cells by inducing the NK cells killing-suppressing receptors TIGIT and CEACAM1. In this study, we show that the incubation of F. nucleatum with primary human natural killer (NK) cells causes the cleavage of the activating receptors CD16, NKp44 and NKp46 by fusolisin. Fusolisin is a fusobacterial outer-membrane, auto-transporter, serine protease and to date the only functional protease found in F. nucleatum. We previously showed that fusolisin is essential for fusobacterial growth in culture. High expression of a functional recombinant fusolisin in E. coli was enabled using codon optimization and replacement of the fusolisin's signal peptide with that of the E. coli OmpA. Recombinant fusolisin was found to cleave the same NK-activating receptors degraded by F. nucleatum. Cleavage of these activating receptors by fusolisin inhibited NK cells activity including killing of tumor cells in-vitro and in-vivo. Our results provide a new bacterial protease-dependent mechanism in which tumors colonized by F. nucleatum are protected from NK cells attack by exploiting fusolisin proteolytic activity. Importantly, our previous and current results suggest that fusolisin might serve to target for tumor-colonized fusobacteria. Citation Format: Amjad Shhadeh, Liat Dassa, Jamal Fahoum, Nicole Haj, Reuven Wiener, Ofer Mandelboim, Gilad Bachrach. Suppression of anti-tumor immunity by the Fusobacterium nucleatum protease fusolisin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5893.