Preclinical studies have suggested that cytotoxic agents and radiation may partly deliver their antitumor activities by activating antitumor immune response. However, the alterations of tumor immune microenvironment including immunosuppressive molecules during chemoradiotherapy and their associations with clinical features and prognosis in rectal cancer have not been thoroughly investigated. Therefore, we investigate the densities of cluster of differentiation 8 (CD8) positive tumor-infiltrating lymphocytes (TILs), CD4+TILs, natural killer cell (NK)-TILs, myeloid-derived suppressor cells (MDSCs), transcription factor forkhead box P3 (FOXP3)+TILs, programmed death ligand-1 (PD-L1), and cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) before and after neoadjuvant chemoradiotherapy (nCRT) in rectal cancer patients to determine their predictive and prognostic effects. We screen 62 rectal cancer patients who underwent nCRT followed by radical surgery. Pretreatment biopsy specimens and posttreatment surgically resected specimens of all patients are retrieved to perform the immunohistochemistry of CD8, CD4, CD56, FOXP3, CD33, CD11b, PD-L1, and CTLA-4. The CD8+TILs and CD4+TILs in post-nCRT resected specimens are significantly higher than that in pre-nCRT biopsy specimens (P= 0.004 and 0.005, respectively). Expressions of MDSC, FOXP3+TILs, and CTLA-4 are relative stable after nCRT. Tumors with high density of CD8+TILs, CD4+TILs, and low MDSC-TILs are more sensitive to nCRT (P= 0.022, 0.022 and 0.005, respectively). High pretreatment CD8+TILs are associated with better disease-free survival and overall survival (P= 0.016 and 0.022, respectively). NK-TILs are detected only in 6 of 62 rectal cancer specimens evaluated. Cell surface PD-L1 positive by tumor cells (1 of 62) and stroma cells (3 of 62) are very low. We may conclude that tumor immunity is activated after nCRT by increased infiltrating CD8+ and CD4+ T cells and relative stable numbers MDSC-TILs, FOXP3+TILs, and coinhibitory molecules. Pre-nCRT CD8+TILs, CD4+TILs, and MDSC-TILs are sensitive predictive marker for response to CRT, and high CD8+TILs are associated with better prognosis.