The role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr- and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.