Many studies have verified the safety of combined radiotherapy and immune checkpoint blockades iation dose or sequencing of combination. We aimed to evaluate the expression and response of PD-1, TIM-3, LAG-3 after neoadjuvant radiotherapy (NRT) and explore the possibility and optimal schedule of combining immunotherapy with radiotherapy in treating rectal cancer. Immunohistochemistry was performed to detect the expression of PD-1, TIM-3, LAG-3, CD8, and CD3. These molecules' expression was detected on the specimens of 76 rectal cancer patients following NRT and 13 of these patients before NRT. The expression of ICBs was assessed by the percentage of positive cells. The levels of PD-1 and immune cells (ICs) LAG-3 in rectal cancer increased after NRT (0% vs. 3%, P = 0.043 and 5% vs. 45%, P = 0.039, respectively). However, TIM-3 in ICs and tumor cells (TCs) were both decreased (80% vs. 50%, P = 0.011, 90% vs. 0%, P = 0.000, respectively). The LAG-3 expression was higher in patients treated with short-course RT than longcourse RT (22.5% vs. 8.0%, P = 0.0440 in ICs; 0% vs. 70%, P < 0.001 in TCs). On the co ntrary, CD8 was higher after long-course RT (15% vs. 8%, P = 0.0146). Interestingly, the level of ICs TIM-3 was low in > eight weeks after long-course RT (P = 0.045). The expressions of PD-1, ICs TIM-3, ICs LAG-3, CD3, and CD8 were associated with the disease-free survival (DFS) in univariate analysis (P = 0.036, 0.008, 0.018, 0.025, and 0.004, respectively). Adjusted by the relevant variables, PD-1 (HR 0.274; 95%