Abstract The immune system can distinguish healthy cells from tumor cells, as the latter expresses tumor associated antigens (TAAs). In the context of CD8+ T cell-mediated immune responses, recognition of TAAs occurs through the presentation of TAAs via major histocompatibility class I (MHC-I) on tumor cells and their interaction with T- cell receptor (TCR) on the CD8+ T cells. Impairing this event will ultimately reduce or annihilate the CD8+ T-cell mediated tumor cytotoxicity. However, reduction or loss of antigen presentation is a frequent and essential mechanism used by tumor cells to escape immune recognition and destruction, including genomic deletion of MHC-I genes, transcriptional suppression of antigen presentation-associated genes, and dysregulation of tumor antigen processing by 26S immunoproteasome. Despite the recent success in immunotherapies based on direct blockade of immune checkpoint proteins (PD-1, CTLA-4 and PD-L1), a majority of patients with solid tumors do not experience durable clinical benefits. The mechanisms of tumor resistance against immune checkpoint blockade (ICB) involve low levels of tumor infiltrating lymphocytes, suppressive immune cells (Tregs, MDSCs and macrophages), and adverse tumor microenvironment. Aberrations in the antigen processing and presentation machinery (APM) genes correlate with poor clinical outcomes. Therefore, therapeutic intervention of tumor antigen presentation would be a promising therapeutic approach to improve responsiveness towards immune checkpoint blockade. In a screen for small-molecule compounds from herbal medicine that potentiate T cell-mediated cytotoxicity, we identified atractylenolide I (ATT-I) that significantly promotes tumor antigen presentation of both human and mouse colorectal cancer cells and thereby enhances the cytotoxic response of CD8+ T cells. In syngeneic mouse colorectal tumor models, treatment of ATT-I in combination with PD-1 antibody markedly enhanced the efficacy of immune checkpoint blockade therapy. We propose that targeting the function of immunoproteasome with ATT-I promotes tumor antigen presentation, empowers T-cell cytotoxicity, and thus elevates the tumor response to immunotherapy. In this application, we will 1) determine the molecular mechanism by which ATT-I promotes antigen processing and presentation in colorectal cancer cells; 2) evaluate the effect of ATT-I treatment on intra-tumor immune cell infiltration, activity of cytotoxic T lymphocytes, and their interaction with tumor microenvironment in colorectal tumors; 3) assess the therapeutic activity of ATT-I in enhancing the efficacy of immune checkpoint blockade immunotherapy (ICB) using genetically engineered mouse models of colorectal cancer and human ex vivo colorectal tumor organoid models.