The WNT-TCF signaling pathway participates in adult tissue homeostasis and repair, and is hyperactive in a number of human diseases including cancers of the colon. Whereas to date there are no antagonists approved for patient use, a potential problem for their sustained use is the blockade of WNT signaling in healthy tissues, thus provoking potentially serious co-lateral damage. Here we have screened a library of plant and microorganism small molecules for novel WNT signaling antagonists and describe withanolide F as a potent WNT-TCF response blocker. This steroidal lactone inhibits TCF-dependent colon cancer xenograft growth and mimics the effects of genetic blockade of TCF and of ivermectin, a previously reported WNT-TCF blocker. However, withanolide F is unique in that it imposes a long-lasting repression of tumor growth, WNT-TCF targets and cancer stem cell clonogenicity after drug treatment. These findings are paralleled by its modulation of chromatin regulators and its alteration of overall H3K4me1 levels. Our results open up the possibility to permanently repress essential signaling responses in cancer cells through limited treatments with small molecules.