〔Abstract〕 Objective To explore the mechanism of bufalin on tumor metastasis mediated by cancer-associated fibroblasts(CAF). Methods The primary CAFs were extracted from the tissues of colon cancer patients, classified into CAF-1 and CAF-2, and their morphology and surface markers were observed under confocal microscopy. Western blot detected the expression of CAF surface markers fibroblast activated protein(FAP), α-smooth muscle actin(α-SMA), and Vimentin(VIM) to confirm the identity of CAF. The CAF conditioned medium was collected, and the interleukin-6(IL-6) secretion level in the conditioned medium was detected by Elisa experiment. HCT116 cells were divided into 2 groups, which was HCT116 group and HCT116 with CAF conditioned medium group. The expression of P-STAT3 and STAT3, proteins related to STAT3 pathway in HCT116, was detected by Western blot. Western blot was used to detect epithelial mesenchymal transformation(EMT) of HCT116 and the changes in the migration and invasion ability of HCT116 were confirmed by scratch experiment and Transwell experiment. Two groups of HCT116 cells, including a control group and a bufalin group, were both treated with CAF-conditioned medium to observe the activation of STAT3 pathway and the changes in migration and invasion capabilities. Results The extracted cells showed a long spindle shape and expressed CAF surface markers FAP, α-SMA and VIM. The Elisa experiment showed that CAF secreted IL-6; Western blot results showed that the conditioned medium of CAF activated the STAT3 signaling pathway of HCT116; Western blot, the scratch experiment and Transwell experiment also found that the migration and invasion ability of HCT116 increased. When bufalin was added, the migration promotion effect of CAF was inhibited. Conclusion CAF activates the STAT3 pathway of tumor cells by releasing IL-6, and promotes the invasion and metastasis of colorectal cancer. Bufalin blocks the process by inhibiting the activation of the STAT3 pathway on tumor cells.