〔Abstract〕 Objective To investigate the effect of cinobufacini on inhibiting colorectal cancer metastasis by regulating the polarization of M2 macrophages. Methods THP-1 was induced into M0 type macrophages. The conditioned medium of HCT116 cells was collected to stimulate M0 type macrophages. The polarization of M2 type macrophages was observed by flow cytometry, real-time quantitative PCR and ELISA experiments. The conditioned medium of M0 type macrophages and HCT116-Mφ cells was collected to stimulate HCT116 cells. The ability of migration and invasion was observed by wound healing assay and Transwell assay. The effect of cinobufacini on the viability of HCT116 cells was detected by CCK-8 assay. The conditioned medium of HCT116 and HCT116+cinobufacini was collected to stimulate M0 type macrophages. The polarization of M2 type macrophages was observed by flow cytometry, real-time quantitative PCR and ELISA experiments. The conditioned media of HCT116-Mφ cells and(HCT116+cinobufacini)-Mφ cells were collected to stimulate HCT116 cells. The changes of migration and invasion ability were observed by wound healing assay and Transwell assay. Results After stimulation of M0 type macrophages in HCT116 cell conditioned medium, the morphology of M0 macrophages turned into fusiform cells, the proportion of CD11b+CD206+cells increased, and the expression of M2 macrophage markers IL-10 and TGF-β increased. The migration and invasion ability of HCT116 cells were significantly enhanced after stimulation in the conditioned medium of HCT116-Mφ cells. After the addition of cinobufacini, not only the polarization proportion of M2 macrophages decreased, but also the metastatic effect mediated by M2 macrophages was inhibited. Conclusion HCT116 cells can induce the polarization of M2 macrophages, while cinobufacini can inhibit the tumor metastasis mediated by M2 macrophages by inhibiting the polarization of M2 macrophages.