The deregulation of the Wnt/β-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/β-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/β-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs β-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/β-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.