〔Abstract〕 Objective To investigate the reversal effect of cinobufagin(CINO) combined with 5-fluorouracil(5-FU) on human colorectal cancer(CRC) drug-resistant cell line HCT15/5-FU, and to clarify the regulatory role of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) pathway in reversing chemoresistance of colorectal cancer. Methods MTT assay was used to detect the changes of drug resistance and drug resistance index, flow cytometry was used to evaluate the apoptosis of cells, scratch test and Transwell assay were used to detect the changes of cell migration and invasion ability. Western blot was used to detect the expressions of epithelial-mesenchymal transition(EMT) related proteins and HIF-1α/VEGF pathway-related proteins. Results Compared with HCT15 cells, the resistance index of HCT15/5-FU was about 8.720. CINO combined with 5-FU could significantly enhance the drug sensitivity of HCT15/5-FU cells, reduce drug resistance index, up-regulate the level of apoptosis, and inhibit cell migration and invasion in a dose-dependent manner. Western blot results showed that CINO combined with 5-FU could inhibit the activity of EMT and HIF-1α/VEGF pathway. Conclusion CINO can reverse 5-FU resistance of colorectal cancerin vitro, and its mechanism may be related to the regulation of the HIF-1α/VEGF pathway and the inhibition of EMT and angiogenesis.