Reversible acetylation of α-tubulin has been implicated in regulating microtubule stability and function. The distribution of acetylated α-tubulin is tightly controlled and stereotypic. Acetylated α-tubulin is most abundant in stable microtubules but is absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts^,. However, the enzymes responsible for regulating tubulin acetylation and deacetylation are not known. Here we report that a member of the histone deacetylase family, HDAC6, functions as a tubulin deacetylase. HDAC6 is localized exclusively in the cytoplasm, where it associates with microtubules and localizes with the microtubule motor complex containing p150^glued (ref. ). In vivo, the overexpression of HDAC6 leads to a global deacetylation of α-tubulin, whereas a decrease in HDAC6 increases α-tubulin acetylation. In vitro, purified HDAC6 potently deacetylates α-tubulin in assembled microtubules. Furthermore, overexpression of HDAC6 promotes chemotactic cell movement, supporting the idea that HDAC6-mediated deacetylation regulates microtubule-dependent cell motility. Our results show that HDAC6 is the tubulin deacetylase, and provide evidence that reversible acetylation regulates important biological processes beyond histone metabolism and gene transcription.