The cytoskeleton of eukaryotic cells undergoes a reorganization in response to intracellular and extracellular cues, which plays an essential role in orchestrating various cell functions including migration, development, differentiation, tissue homeostasis, contractility, proliferation, gene expression, cancer cell invasion, and airway/vascular remodeling. Acetylation occurs on the cytoskeletal components, such as microtubules, actin, and vimentin, which regulate cellular functions. Moreover, remodeling of the cytoskeleton is regulated by acetylation and deacetylation of regulatory proteins, including adapter proteins and protein kinases. Therefore, protein acetylation and deacetylation are critical mechanisms for cytoskeletal reorganization in response to changes of intracellular and extracellular environments.