Histone deacetylase 6 (HDAC6) contains tandem catalytic domains and a ubiquitin-binding zinc finger and displays deacetylase activity toward acetylated microtubules. Here we show that unlike its orthologs from Caenorhabditis elegans, Drosophila, and mouse, human HDAC6 possesses a tetradecapeptide repeat domain located between the second deacetylase domain and the C-terminal ubiquitin-binding motif. Related to this structural difference, the cytoplasmic localization of human, but not murine, HDAC6 is resistant to treatment with leptomycin B (LMB). Although it is dispensable for the deacetylase and ubiquitin binding activities of human HDAC6, the tetradecapeptide repeat domain displays acetyl-microtubule targeting ability. Moreover, it forms a unique structure and is required for the LMB-resistant cytoplasmic localization of human HDAC6. Besides the tetradecapeptide repeat domain, human HDAC6 possesses two LMB-sensitive nuclear export signals and a nuclear localization signal. These results thus indicate that the cytoplasmic localization for murine and human HDAC6 proteins is differentially regulated and suggest that the tetradecapeptide repeat domain serves as an important sequence element to stably retain human HDAC6 in the cytoplasm.