Background <p>Recent studies have revealed that CD8⁺ T cells can be activated via genetic upregulation of HIF-1α, thereby augmenting antitumor effector functions. HIF-1α upregulation can be attained by inhibiting HIF-prolyl hydroxylase (HIF-PH) under normoxic conditions, termed pseudohypoxia. This study investigated whether pseudohypoxia induced by HIF-PH inhibitors suppresses Microsatellite stable (MSS) colorectal cancer (CRC) by affecting tumor immune response.</p> Methods <p>The HIF-PH inhibitors Roxadustat and Vadadustat were utilized in this study. In vitro, we assessed the effects of HIF-PH inhibitors on human and murine colon cancer cell lines (SW480, HT29, Colon26) and murine T cells. In vivo experiments were performed with mice bearing Colon26 tumors to evaluate the effect of these inhibitors on tumor immune responses. Tumor and spleen samples were analyzed using immunohistochemistry, RT-qPCR, and flow cytometry to elucidate potential mechanisms.</p> Results <p>HIF-PH inhibitors demonstrated antitumor effects in vivo but not in vitro. These inhibitors enhanced the tumor immune response by increasing the infiltration of CD8⁺ and CD4⁺ tumor-infiltrating lymphocytes (TILs). HIF-PH inhibitors induced IL-2 production in splenic and intratumoral CD4⁺ T cells, promoting T cell proliferation, differentiation, and immune responses. Roxadustat synergistically enhanced the efficacy of anti-PD-1 antibody for MSS cancer by increasing the recruitment of TILs and augmenting effector-like CD8⁺ T cells.</p> Conclusion <p>Pseudohypoxia induced by HIF-PH inhibitors activates antitumor immune responses, at least in part, through the induction of IL-2 secretion from CD4⁺ T cells in the spleen and tumor microenvironment, thereby enhancing immune efficacy against MSS CRC.</p>