Colon cancer is one of the most common malignancies. Although there has been great development in treatment regimens over the last few decades, its prognosis remains poor. There is still a clinical need to find new drugs for colon cancer. Evodiamine (Evo) is a quinolone alkaloid extracted from the traditional herbal medicine plant Evodia rutaecarpa. In the present study, CCK-8, flow cytometry, reverse transcription quantitative polymerase chain reaction, western blot analysis and a xenograft tumor model were used to evaluate the anti-cancer activity of Evo in human colon cancer cells and determine the possible mechanism underlying this process. It was revealed that Evo exhibited prominent anti-proliferation and apoptosis-inducing effects in HCT116 cells. Bone morphogenetic protein 9 (BMP9) was notably upregulated by Evo in HCT116 cells. Exogenous BMP9 potentiated the anti-cancer activity of Evo, and BMP9 silencing reduced this effect. In addition, HIF-1α was also upregulated by Evo. The anticancer activity of Evo was enhanced by HIF-1α, but was reduced by HIF-1α silencing. BMP9 potentiated the effect of Evo on the upregulation of HIF-1α, and enhanced the antitumor effect of Evo in colon cancer, which was clearly reduced by HIF-1α silencing. In HCT116 cells, Evo increased the phosphorylation of p53, which was enhanced by BMP9 but reduced by BMP9 silencing. Furthermore, the effect of Evo on p53 was potentiated by HIF-1α and reduced by HIF-1α silencing. The present findings therefore strongly indicated that the anticancer activity of Evo may be partly mediated by BMP9 upregulation, which can activate p53 through upregulation of HIF-1α, at least in human colon cancer.