The type B trichothecene mycotoxin deoxynivalenol (DON), colloquially known as "vomitoxin", is the most commonly detected trichothecene in cereal-based foods, exerting acute and chronic toxic effects on animals and causing serious safety-related concerns. At the cellular and molecular levels, DON can inhibit macromolecule synthesis by binding to ribosomes and may disrupt cell proliferation, differentiation and apoptosis. However, the molecular mechanisms underlying the cytotoxicity of DON remain to be determined. Here, we identified β-catenin, the key signal transducer of the Wnt cascade, as a novel target of DON by quantitative real-time PCR and Western blot analysis in human embryonic kidney 293T and colorectal SW480 cells treated with DON at half IC50 (50 ng/mL for HEK293T and 1 μg/mL for SW480). Further analysis showed that neither posttranslationalmodification nor nuclear accumulation of β-catenin was affected post DON treatment. Moreover, we found that the β-catenin-dependent canonical Wnt signaling pathway, which regulates many biological processes during embryonic development and adult tissue homeostasis, was involved in DON-induced inhibition of cell proliferation. Then, we determined that the β-catenin/c-Myc axis was essential for this process, as the DON-induced inhibition of cell proliferation was efficiently rescued by restoration of β-catenin or c-Myc levels. Our results advance the current understanding of the molecular toxicological mechanism of DON and provide a new perspective on strategies for the prevention and control of mycotoxins.