Microtubules (MTs) provide mechanical strength to cardiomyocytes and dysregulation of MT network is implicated in cardiac diseases. MT acetylation mediates mechanotransduction, provides structural flexibility to cardiomyocytes and protects against proteinopathy-induced cardiac failure. MT acetylation is exclusively catalyzed by α-TAT1, whose only known substrate is α-tubulin in polymerized MTs. However, little is known about how α-TAT1 itself is regulated. Here we report that intracellular spatial localization of α-TAT1 mediates MT acetylation. Specifically, we identified a conserved signal motif in the intrinsically disordered C-terminus of α-TAT1, consisting of three competing regulatory elements - nuclear export, nuclear import and cytosolic retention. Inhibition of Exportin 1-mediated nuclear export induced nuclear accumulation of α-TAT1 and loss of MT acetylation. We found that α-TAT1 nuclear localization is inhibited by CDKs, CK2 and PKA kinases, pharmacological inhibition of which increased nuclear localization of α-TAT1 and inhibited MT acetylation. We identified a critical phosphoThreonine (T 322 ) that binds to 14-3-3 proteins (β, γ, ε and ζ isoforms) downstream of kinases and mediates cytosolic retention of α-TAT1. Inhibition of 14-3-3 proteins also increased nuclear accumulation of α-TAT1. Fibroblastic cells expressing a phosphodeficient α-TAT1 (T322A) show defects in DNA damage response and increased cell proliferation, which may be pertinent to cardiac hypertrophy. Based on these observations, we developed an optogenetic tool, named optoATAT1, which rapidly and reversibly shuttled from the nucleus to the cytosol on blue light stimulation. HeLa cells expressing optoATAT1 exposed to light showed increased MT acetylation unlike those kept in dark, validating the tool. In summary, we have identified a novel role for the C-terminal region of α-TAT1 in regulating its function through dynamic intracellular localization downstream of kinases and 14-3-3 proteins. We have identified multiple pharmacological agents to modulate MT acetylation through spatial regulation of α-TAT1. We have also developed an optogenetic tool to control MT acetylation that will help in elucidating the role of MT acetylation in disease states.