ETHNOPHARMACOLOGICAL RELEVANCE: Huachansu (HCS), a traditional Chinese medicine (TCM), has been used as an adjuvant therapy for colorectal cancer (CRC). However, its underlying mechanisms for combating CRC require further investigation. AIM OF THIS STUDY: To comprehensively evaluate the anti-CRC effects of HCS and elucidate its underlying mechanisms, with a focus on elucidating the key pathways and targets involved. MATERIALS AND METHODS: A series of cell experiments and xenograft tumor models were used to evaluate the inhibitory effects of HCS. The key components and potential targets of HCS against CRC were identified through network pharmacology and molecular docking. To further investigate the mechanisms, transcriptomics and proteomics were integrated, and the findings were supported by systematic pharmacological validation. Finally, the efficacy of HCS was further confirmed in CRC Patients-derived organoid and orthotopic models. RESULTS: HCS could inhibit proliferation, disrupt the cell cycle, induce apoptosis of CRC cells, and suppress the growth of CRC xenograft tumors. Then eight components and six proteins (PIK3CA, CTNNB1, TP53, AKT1, CCND1, and CDH1) were identified as critical for HCS's anti-CRC activity. Notably, HCS inhibited the PI3K/AKT signaling pathway and glycolysis in CRC cells, with these findings validated in both in vitro and in vivo models. Additionally, HCS reduced growth in CRC patient-derived organoids and orthotopic models. CONCLUSION: This study elucidates the mechanisms of HCS to combat CRC, offering a valuable reference for future clinical applications. It also presents a distinctive strategy for exploring TCM formulations' active components and effective mechanisms.