Immune checkpoint inhibitors (ICIs) are potentially able to be strong strategy to treat the Head and Neck Cancer, however they lack enough their efficacy, so the search for factors that can improve their efficacy is needed. The gut microbiota and its metabolites have been found to play an important role via modification tumor microenvironment with the treatment of ICIs. It was recently reported that inosine, one of the gut bacterial metabolites, modulates cancer immunity, however the detail of the mechanism is still unclear. In the present study, we attempt to clarify the direct effects of inosine to cancer cells and immune cells in vitro. In the equilibrium phase of cancer immunoediting, it is important to make the immune side dominant in order to control cancer, thus inosine was added directly to co-cultures of T cells and cancer cells reproducing the equilibrium phase between cancer and immunity. These cells are harvested and divided into cancer cell and immune cell subsets followed by immunological analysis. We also investigated the in vivo efficacy of inosine utilizing BALB/c mice with CT26 murine colon cancer model. As a result, it was found that there is an optimal concentration of inosine that inhibits cancer cell growth in co-cultures of T cells and cancer cells. The anti-tumor effect differs depending on the concentration and route of administration in a tumor-bearing mouse model. In the future, we will find the conditions for inhibiting tumor growth and activating T cells, which will lead to the development of tumor immunotherapy against Head and Neck Cancer using inosine.