Acetylation is a well-studied post-translational modification (PTM) of tubulin. Acetylated tubulin is present in the centrioles, primary cilia, and flagella, which contain long-lived stable microtubules. Tubulin acetylation plays an important role in cellular activities including cell polarity, cell migration, vesicle transport, and cell development. Cryo-electron microscopy reconstructions have revealed conformational changes in acetylated tubulin, revealing a reduction in intermonomer interactions among tubulins and an increase in microtubule elasticity. The kinetics of conformational changes in acetylated tubulin may elucidate microtubule functions in these cellular activities. Abnormal tubulin acetylation has been implicated in neurodegenerative disorders, ciliopathies, and cancers. Thus, it is important to elucidate the mechanisms underlying tubulin acetylation and its effects on cellular activity to understand these diseases and to design potential therapeutic strategies. This review discusses the cellular distribution and dynamics of acetylated tubulin and its role in regulating cellular activities.