AbstractThe optimum fractionation of radiation to combine with immune checkpoint blockade is controversial. This study aimed to investigate the fractionated radiation to maximize immunity during combination therapy. To evaluate the abscopal effect, C57BL/6 hPD‐1 knock‐in mice bearing two syngeneic contralateral MC38 murine colon cancer tumors were treated with four distinct regimens of radiotherapy. Three fractions of 8 Gy were chosen as the optimal fractionation to combine with anti‐PD‐1 as the optimal fractionation for maximizing immunity. Anti‐PD‐1 administration enhanced both local and systemic antitumor immunity in a cytotoxic T cell–dependent manner. Meanwhile, the spleen exhibited decreased myeloid‐derived suppressor cells (MDSCs) under combination treatment. Furthermore, RNA‐sequencing revealed significantly increased tumor necrosis factor (TNF) receptors and cytokines associated with lymphocyte infiltration in the combining group. Here we demonstrate that the hypofractionation of 8 Gy × 3f was the optimum‐fractionated dosage to maximize immunity, and the combination of anti‐PD‐1 showed promising results in boosting abscopal effect. Underlying mechanisms may include the activation of T cells and the reduction of MDSCs, which is achieved through the action of TNF and related cytokines. This study indicates a radioimmunotherapy dosage painting method that can be developed to overcome present limitations in tumor immunosuppression.