Alkaloids derived from natural products have been traditionally used to treat various diseases, including cancers. Rutaecarpine (<b>1</b>), a β-carboline-type alkaloid obtained from <i>Evodia rutaecarpa</i>, has been previously reported as an anti-inflammatory agent. Nonetheless, its anticancer activity and the underlying molecular mechanisms remain to be explored. In the procurement of Wnt/β-catenin inhibitors from natural alkaloids, <b>1</b> was found to exhibit activity against the Wnt/β-catenin-response reporter gene. Since the abnormal activation of Wnt/β-catenin signaling is highly involved in colon carcinogenesis, the antitumor activity and molecular mechanisms of <b>1</b> were investigated in colorectal cancer (CRC) cells. The antiproliferative activity of <b>1</b> was associated with the suppression of the Wnt/β-catenin-mediated signaling pathway and its target gene expression in human CRC cells. <b>1</b> also induced G<sub>0</sub>/G<sub>1</sub> cell cycle arrest and apoptotic cell death, and the antimigration and anti-invasion potential of <b>1</b> was confirmed through epithelial-mesenchymal transition biomarker inhibition by the regulation of Wnt signaling. The antitumor activity of <b>1</b> was supported in an Ls174T-implanted xenograft mouse model via Wnt target gene regulation. Overall, these findings suggest that targeting the Wnt/β-catenin signaling pathway by <b>1</b> is a promising therapeutic option for the treatment of human CRC harboring β-catenin mutation.