Although significant progress has been made in understanding the importance of Wnt signaling in the initiation of colorectal cancer, less is known about responses that accompany the reversal of oncogenic Wnt signaling. The aim of this study was to analyze in vivo and in vitro responses to an ideal Wnt pathway inhibitor as a model for the therapeutic targeting of the pathway. A tetracycline-inducible transgenic mouse model expressing truncated beta-catenin (DeltaN89beta-catenin) that exhibited a strong intestinal hyperplasia was analyzed during the removal of oncogenic beta-catenin expression both in 3D crypt culture and in vivo. Oncogenic Wnt signaling was rapidly and completely reversed. The strongest inhibition of Wnt target gene expression occurred within 24 h of doxycycline removal at which time the target genes Ascl2, Axin2 and C-myc were downregulated to levels below that in the control intestine. In vitro, the small molecule Wnt inhibitor CCT036477 induced a response within 4 h of treatment. By 7 days following doxycycline withdrawal, gene expression, cell proliferation and tissue morphology were undistinguishable from control animals.In conclusion, these results demonstrate that the reversal of Wnt signaling by inhibitors should ideally be studied within hours of treatment. The reversible system described, involving medium throughput in vitro approaches and rapid in vivo responses, should allow the rapid advance of early stage compounds into efficacy models that are more usually considered later in the drug discovery pipeline.Oncogene advance online publication, 2 April 2012; doi:10.1038/onc.2012.103.