ETHNOPHARMACOLOGICAL RELEVANCE: Pien Tze Huang (PZH) is a traditional medicinal formula consisted of four traditional Chinese medicines (TCMs) including Panax notoginseng (Burk.) F. H. Chen, Snake Gall, Calculus Bovis and Moschus, with clinical efficacy against Colorectal Cancer (CRC). However, the molecular and functional mechanisms underlying this efficacy are not fully elucidated. AIMS OF THE STUDY: This study aimed to assess the impact of PZH on CRC cancer stem cells (CSCs), and evaluate the coordination effect of PZH on T cell-mediated anti-CRC with patient-derived autologous T cell co-culture. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC) was used to identify the main components of PZH. CCK8 and spheroid formation assays were conducted for assessing cell viability and stemness function. Western blot, immunofluorescence and immunohistochemistry were used to evaluate CSC markers and PD-L1 expression. T cell successful expansion was validated by flow cytometry. Co-culture assay was conducted to explore the activation effect of PZH on T cells. The potential mechanism of PZH in CRC was identified with transcriptomics sequencing and network pharmacology analysis. RESULTS: PZH reduced cell viability and spheroid formation ability in CRC, and suppressed the expression of CSC markers - LGR5, DCLK1, and CD133. Moreover, PZH enhanced T cell-mediated cytotoxicity against CRC cells by decreasing the expression of PD-L1. Furthermore, PZH with anti-PD-1 immunotherapy enhancing antitumor efficacy and increasing CD8 CONCLUSIONS: PZH enhances T cell-mediated killing by inhibiting the expression of CRC stem cell markers and PD-L1, which warrant further investigation and clinical applications.