The present invention relates to an anti-cancer regulatory response and immunotherapy by Mycobacterium paragordonae (M. paragordonae). Mycobacterium paragordonae according to an aspect has cancer cytotoxicity mediated by natural killer cells or T cells as well as cytotoxicity against cancer cells, and inhibits activation of cancer cytotoxicity regulatory T cells of macrophages or dendritic cells to induce an immune response, thereby inhibiting tumor formation and thus being useful for a cancer immunotherapeutic agent.