PURPOSE/BACKGROUND: Radiotherapy has been recently reported to boost the therapeutic response of immune checkpoint blockade (ICB); however, few studies have focused on programmed cell death-ligand 1 (PD-L1) expression in locally advanced rectal cancer (LARC) patients who receive preoperative neoadjuvant chemoradiotherapy (neoCRT). The aim of the present study was to investigate the PD-L1 expression status and CD8+ intra-tumoral infiltrating lymphocytes (TILs) before and after neoCRT and its association with clinicopathological characteristics in rectal cancer. MATERIALS AND METHODS: Immunostainings of PD-L1 and CD8+ TILs were performed in 112 pair-matched LARC patients treated by neoCRT. Tumor PD-L1 expression and CD8+ TILs within the tumor microenvironment before and after neoCRT were evaluated via immunohistochemistry. RESULTS: High tumor PD-L1 expression was significantly increased from 50 to 63%, and high CD8+ TILs counts were also slightly increased from 32 to 35% after neoCRT treatment. High tumor PD-L1 before and after neoCRT was associated with improved disease-free survival (DFS, pre-neoCRT: p = 0.003 and post-neoCRT: p = 0.003) and overall survival (OS, pre-neoCRT: p = 0.045 and post-neoCRT: p = 0.0001). High CD8+ TILs before neoCRT was associated with improved DFS (p = 0.057), and it was significantly associated with improved DFS after neoCRT (p = 0.039). Patients with high tumor PD-L1 and CD8+ TILs before and after neoCRT were significantly associated with improved DFS (pre-neoCRT: p = 0.004 and post-neoCRT: p = 0.006). CONCLUSION: The present results provide evidence that tumor PD-L1 expression and recruitment of CD8+ TILs within the tumor microenvironment were increased by neoCRT treatment. Tumor PD-L1 and CD8+ TILs are prognostic biomarkers for the survival of LARC patients treated with neoCRT.