To evaluate the impact of hypofractionated radiotherapy (Hypo-RT) with different interfraction intervals on tumor growth, immune response, and synergistic effects with anti-PD-1 immunotherapy.The mouse MC38 colon cancer model was utilized. Various radiation regimens were designed to investigate the effects of fraction interval and fraction size on tumor growth, immune mobilization, and combination effects with anti-PD-1 immunotherapy.For a fixed-dose experiment, the 6 × 5 Gy for every other day (qod) regimen demonstrated an equivalent effect on tumor growth compared to the 6 × 5 Gy once daily (qd) regimen, while the 6 × 5 Gy for twice weekly (biw) regimen failed to inhibit tumor growth. Both qod and biw regimens induced an enhanced immune response, unlike the qd regimen. For a fixed biologically equivalent dose experiment, 6 × 5 Gy qod, 4 × 7 Gy biw, and 2 × 11 Gy once weekly (qw) regimens exhibited similar tumor suppression to the 12 × 3 Gy qd regimen. The long-interval Hypo-RT regimens significantly mobilized host immunity, whereas 12 × 3 Gy qd did not. The peripheral and intratumoral T cells increased as the fraction interval and size increased. All Hypo-RT regimens combined with anti-PD-1 immunotherapy demonstrated higher intratumoral CD8 + T cells and more effective tumor growth delay compared to the 12 × 3 Gy qd regime.The current study suggested that a prolonged inter-fraction interval with an increased fraction size in Hypo-RT may be a promising option to balance the therapeutic effect on tumor and immune activation.Copyright © 2024. Published by Elsevier B.V.