Estrogen regulation of epithelial ion secretion and absorption exhibits sexual dimorphism and increased potency in the female estrous cycle. The molecular target of estrogen’s anti-secretory actions in the intestine is the KCNQ1:KCNE3 channel. We have recently discovered that this K+ channel is complexed with β-catenin at the plasmamembrane of colonic epithelial cells and the bidirectional interactions between KCNQ1:β-catenin modulate colorectal cancer (CRC) epithelial to mesenchymal transition (1). We investigated the molecular mechanisms regulating KCNQ1:β-catenin interactions and their effects on CRC cell differentiation, proliferation and invasion. Molecular and pharmacological approaches were used to determine the influence of KCNQ1 expression on the Wnt/β-catenin signaling pathway and epithelial-to-mesenchymal transition (EMT) in human CRC cell lines of varying stages of differentiation. The expression of KCNQ1 was lost with increasing mesenchymal phenotype in poorly differentiated CRC cell lines as a consequence of repression of the KCNQ1 promoter by β-catenin:TCF4. In well-differentiated epithelial CRC cell lines, KCNQ1 was localized to the plasmamembrane in a complex with β-catenin and E-cadherin. The co-localization of KCNQ1 with adherens junction proteins was lost with increasing EMT phenotype. ShRNA knock-down of KCNQ1 caused a re-localization of β-catenin from the plasma membrane and a loss of epithelial phenotype in CRC spheroids. Over-expression of KCNQ1 trapped β-catenin at the plasmamembrane, induced a patent lumen in poorly differentiated CRC spheroids and slowed CRC cell invasion. The KCNQ1 ion channel inhibitor chromanol 293B caused membrane depolarization, redistribution of β-catenin into the cytosol, a reduced transepithelial electrical resistance and stimulated CRC cell proliferation. Analysis of human primary CRC tumour patient databases showed a positive correlation between KCNQ1:KCNE3 expression and disease-free survival in females but not in males. We conclude that the KCNQ1 ion channel is a novel estrogen target gene in colon cancer and a regulator of the Wnt/β-catenin pathway. The estrogen modulation of KCNQ1:KCNE3 underlies the sexual dimorphism of the hormone’s protective effects in CRC female patient survival through repression of Wnt/β-catenin, CRC cell proliferation, EMT and tumourigenesis.