AbstractChelerythrine chloride (CHE) is a benzodiazepine alkaloid derived from natural herbs with significant anti‐tumor and anti‐inflammatory activities. However, the exact role and underlying mechanisms of CHE in colorectal cancer (CRC) remain unclear. Therefore, this study is aimed to investigate the influence of CHE on the progression of CRC. Cell Counting Kit‐8 assay (CCK‐8), transwell, apoptosis rate, cell cycle distribution, reactive oxygen species (ROS), and colony formation determined the anti‐proliferative activity of CHE in CRC cell lines. Transcriptome sequencing and western blot were used to explore the mechanism. Finally, H&E staining, Ki67, TUNEL, and immunofluorescence were conducted to verify the anti‐CRC activity and potential mechanisms of CHE in vivo. CHE had a prominent inhibitory effect on the proliferation of CRC cells. CHE induces G1 and S phase arrest and induces cell apoptosis by ROS accumulation. Cancer‐associated fibroblasts (CAFs) play a key role in CRC metastasis. Then, this study found that CHE regulates WNT10B/β‐catenin and TGFβ2/Smad2/3 axis, thereby decreasing the expression of α‐SMA, which is a maker of CAFs. Taken together, CHE is a candidate drug and a potent compound for metastatic CRC, which can intervene CAFs in a dual pathway to effectively inhibit the invasion and migration of cancer cells, which can provide a new choice for future clinical treatment.