In epithelial cells, the apical junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell-surface polarity by forming a fence that prevents lateral movement and diffusion of proteins and lipids between the apical and basolateral PM and holds the epithelial monolayer intact through cell–cell contacts. Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton. Recent findings also point to the involvement of the microtubule (MT) system. Here we show the first evidence that in polarized epithelial MDCK cells, ARF-like protein 2 (ARL2) and -tubulin cofactor D, known to be involved in MT dynamics, have a role in disassembly of the AJC followed by cell dissociation from the epithelial monolayer, which is not dependent on MT depolymerization. In addition, we show that -tubulin cofactor D is partially localized to the lateral PM through its 15 C-terminal amino acids and intact MTs. ARL2 inhibited -tubulin cofactor D-dependent cell dissociation from the monolayer and AJC disassembly. To our knowledge, this is the first evidence that -tubulin cofactor D plays a role in cells independent of its presumed role in folding tubulin heterodimers. We conclude that ARL2 and -tubulin cofactor D participate in AJC disassembly and epithelial depolarization.—Shultz, T., Shmuel, M., Hyman, T., and Altschuler, Y. -tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure.