Detyrosination of α-tubulin seems to be conserved in all eukaryotes. However, its biological function in plants has remained obscure. A conserved C-terminal tyrosine is removed by a still unidentified tubulin–tyrosine carboxypeptidase (TTC) and can be religated by a tubulin–tyrosine ligase (TTL). To obtain insight into the still elusive biological function of this detyrosination–tyrosination cycle, the effects of the TTC inhibitor parthenolide were analysed in BY-2 tobacco cells. Parthenolide caused a depletion of detyrosinated α-tubulin, whereas the level of tyrosinated tubulin was elevated. This biochemical effect was accompanied by growth inhibition in cycling BY-2 cells and alteration of microtubule-dependent events that define division and expansion geometry such as cell plate alignment or axial expansion. Furthermore, parthenolide triggered an apoplastic alkalinization indicative of activation of defence-related calcium influx channels. At the same time, parthenolide promoted the association of the plant-specific kinesin KCH with cortical microtubules. These observations are integrated into a working model, where detyrosination acts as signal to modulate the binding of kinesin motors involved in structural and sensory functions of the microtubular cytoskeleton.