Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved and the most prominent substrate is tubulin, the microtubule (MT) building block. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes and be involved in cell cycle and cell proliferation.