BACKGROUND: Colorectal cancer (CRC) holds the third position in terms of incidence and ranks behind lung cancer in terms of mortality worldwide. Yuanhuacine, one of the main active ingredients of genkwa flos, has demonstrated promising application prospects in the field of cancer treatment. However, its underlying mechanism against CRC has not been fully clarified. PURPOSE: This study aimed to investigate anti-tumor activity of yuanhuacine and clarify its underlying mechanism in CRC. METHODS: CRC HCT116, HT-29, Caco-2, SW480, and LS174T cells were used to assess the in vitro anti-tumor activity of yuanhuacine by cell viability, proliferation, apoptosis, cycle distribution, migration, and colony formation assays. Meanwhile, an HT-29 xenograft mouse model was successfully constructed to investigate the anti-tumor effect of yuanhuacine in vivo. Transcriptomic assay and network pharmacology were applied to explore the underlying mechanism of yuanhuacine in combating CRC, which was further verified by quantitative reverse transcription polymerase chain reaction, western blot. The interaction of yuanhuacine with protein was performed by molecular docking, molecular dynamics simulation, and cell thermal shift assays. RESULTS: Yuanhuacine significantly induced apoptosis and reduced viability of CRC cells with IC CONCLUSIONS: Yuanhuacine is a promising candidate compound in combating CRC by inhibiting proliferation of CRC cells. The major underlying mechanism involves regulating PLK1, which results in G2/M phase arrest.