Cell polarity is essential for spatially regulating of physiological processes in metazoans by which hormonal stimulation‒secretion coupling is precisely coupled for tissue homeostasis and organ communications. However, the molecular mechanisms underlying epithelial cell polarity establishment remain elusive. Here, we show that septin cytoskeleton interacts with catenin complex to organize a functional domain to separate apical from basal membranes in polarized epithelial cells. Using polarized epithelial cell monolayer as a model system with transepithelial electrical resistance as functional readout, our studies show that septins are essential for epithelial cell polarization. Our proteomic analyses discovered a novel septin‒catenin complex during epithelial cell polarization. The functional relevance of septin‒catenin complex was then examined in three-dimensional (3D) culture in which suppression of septins resulted in deformation of apical lumen in cysts, a hallmark seen in polarity-deficient 3D cultures and animals. Mechanistically, septin cytoskeleton stabilizes the association of adherens catenin complex with actin cytoskeleton, and depletion or disruption of septin cytoskeleton liberates adherens junction and polarity complexes into the cytoplasm. Together, these findings reveal a previously unrecognized role for septin cytoskeleton in the polarization of the apical‒basal axis and lumen formation in polarized epithelial cells.