The cytoskeleton is conserved throughout the eukaryotic lineage and consists of a complex dynamic network mainly composed of three distinct polymers: microtubules (MTs), actin filaments, and intermediate filaments. MTs are polymers of α/β-tubulin heterodimers, playing a myriad of distinct cellular functions and are the main components of complex structures like the mitotic spindle, cilia, and centrioles. Post-translational modifications (PTMs) regulate the function and increase the complexity of the α/β-tubulin heterodimer pools. One of the PTMs that has been extensively studied is the acetylation of lysine 40 (K40) on α-tubulin, which specifically occurs inside the MT lumen.Acetylation plays a crucial role in controlling the stability and function of MTs, in response to signals from within and outside the cell. It impacts the cytoplasm's 3D arrangement and important cellular activities like intracellular transport, cell division, polarity, and migration. Recent research has also emphasized the significance of this PTM in regulating the mechanical properties of MTs and cellular sensing. The levels and activity of MT acetyltransferases and deacetylases are tightly regulated through various transcriptional, post-transcriptional, and post-translational mechanisms, including miRNAs, phosphorylation, protein-protein interactions, and regulated localization between the nucleus and cytoplasm. These regulatory processes involve components of diverse signaling pathways, and their deregulation has been implicated in numerous diseases, including neurological disorders, cancer, and cardiac conditions.