Tubulin, the most abundant axonemal protein, is extensively modified by several highly conserved post-translational mechanisms including acetylation, detyrosination, glutamylation, and glycylation. We discuss the pathways that contribute to the assembly and maintenance of axonemal microtubules, with emphasis on the potential functions of post-translational modifications that affect tubulin. The recent identification of a number of tubulin modifying enzymes and mutational studies of modification sites on tubulin have allowed for significant functional insights. Polymeric modifications of tubulin (glutamylation and glycylation) have emerged as important determinants of the 9 + 2 axoneme assembly and motility.