BACKGROUND: The anti-tumor effects of Traditional Chinese Medicine has been recognized in regulating the tumor microenvironment. Jianpi Jiedu Recipe (JPJDR) is clinically effective in enhancing the chemotherapy efficacy in Colorectal Cancer (CRC) treatment and significantly improved the quality of life of CRC patients. However, its therapeutic mechanism remains to be investigated. MATERIAL AND METHODS: The active compounds of each herb in JPJDR were obtained from the HIT2.0 and HERB databases supported by evidence in literature. Single-cell RNA sequencing data of CRC were obtained from published studies (PMID: 32451460, 32103181, and 32561858). Pathway enrichment was analyzed using the reactome database, and gene correlation analysis was performed using the corrplot R software package. Gene expression regulated by JPJDR was verified by RT-qPCR. RESULTS: Ye Pu Tao Teng, Ba Yue Zha, Huang Qi, Bai Zhu, Yi Yi Ren and Dang Shen contained 4,7, 18, 26,3 and 30 compounds, respectively, and they target to 38, 7, 262, 309, 50, 54 genes, respectively. JPJDR is involved in a range of immune-related biological processes, including cytokine signaling, cell proliferation, apoptosis and cellular senescence. JPJDR regulates various cell types in the CRC microenvironment, including malignant CRC cells, immune cells (including CD4+ T cell, CD8+ T cell, B cell, plasma cell, mast cell, and myeloid cell), and stromal cells (including fibroblast, pericyte, enteric glial cell, and endothelial cell). We confirmed that JPJDR significantly downregulated the expression of HMGB1 and MT2A in CRC. CONCLUSIONS: JPJDR regulates a range of cell types in the CRC microenvironment, including malignant CRC, immune cells and stromal cells. Downregulation of HMGB1 and MT2A might be the important mediators for JPJDR to modulate the CRC microenvironment.