Objective: This study aimed to investigate the association between tumor mutational burden (TMB), tumor-infiltrating immune cells (TICs) and clinicopathological features, and their clinical implications in the progression and prognosis of colorectal cancer (CRC). Methods: Data relating to CRC were downloaded from The Cancer Genome Atlas (TCGA) database, including genetic mutation files, RNA-seq data, and clinical information. TMB value and percentage of TICs of each tumor sample were calculated separately using R language; then correlation analysis was performed between these indices, clinicopathological parameters, and the 5-year overall survival rate. Based on the median TMB values, tumors samples were divided into a high TMB group (n=224) and a low TMB group (n=246). Five-year overall survival rates were compared between the 2 groups. Differentially expressed genes (DEGs) and TICs between high-TMB and low-TMB groups were screened and the potential immune mechanism and functions were identified by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA). Results: Based on TCGA data, CRC patients >65 years had higher TMB values; but tumors with lymph node invasion and vascular invasion had a lower TMB values (P<0.001). Compared with the low-TMB group, CRC patients in the high-TMB group had shorter 5-year overall survival rate (P<0.05). A total of 116 DEGs involved in signaling pathway of drug metabolism and leukocyte transendothelial migration, and four special types of TICs, including CD4⁺ T cells, follicular helper T cells, M0 and M1 macrophages, were identified. Conclusion: TMB is an important prognostic and immunotherapeutic index in CRC, which can interact with TICs to modulate the tumor microenvironment of CRC and therefore impact the prognosis of CRC and the efficacy of immunotherapy.