Epithelial integrity is lost upon cancer progression as cancer cells detach from the primary tumor site and start to invade to the surrounding tissues. Invasive cancers of epithelial origin often express altered levels of TRP-family cation channels. Upregulation of TRPV6 Ca2+-channel has been associated with a number of human malignancies and its high expression in breast cancer has been linked to both proliferation and invasive disease. The mechanisms behind the potential of TRPV6 to induce invasive progression have, however, not been well elucidated. Here we show that TRPV6 is connected to both E-cadherin-based adherens junctions and intracellular cytoskeletal structures. Loss of TRPV6 from normal mammary epithelial cells led to disruption of epithelial integrity and abnormal 3D-mammo sphere morphology. Furthermore, expression level of TRPV6 was tightly linked to the levels of common EMT markers, suggesting that TRPV6 may have a role in the mesenchymal invasion of breast cancer cells. Thus, either too low or too high TRPV6 levels compromise homeostasis of the mammary epithelial sheets and may promote the progression of pathophysiological conditions.