Histone deacetylase inhibitors (HDACI) are powerful antiproliferative drugs, and are currently undergoing clinical trials as antitumor agents. It would be valuable for both cancer therapy and our knowledge of basic cellular processes to understand the mechanisms by which HDACIs block cell proliferation. Most current models postulate that HDACIs allow the reexpression of tumor suppressor genes silenced in cancer cells. However, other mechanisms, distinct from transcription regulation, may participate in HDACI antiproliferative properties. We report that HDACI treatment induces premature sister chromatid separation in cells in which the mitotic spindle assembly checkpoint (SAC) has already been activated. This effect was transcription-independent. In addition, HDACI-treated mitotic cells displayed SAC inactivation characteristics, including anaphase-promoting complex/cyclosome target degradation, cyclin-dependent kinase 1 inactivation, histone H3 dephosphorylation, and loss of the SAC component MAD2 from the kinetochore. Thus, HDAC inhibition renders the SAC ineffective. Our findings help elucidate the molecular mechanisms of proliferative cell death induced by HDACI treatment and may allow new HDACI-based preclinical and clinical trial protocols to be redesigned so as to target mitosis.