Protein acetylation and deacetylation play key roles in multiple physiological functions. Histone deacetylase 3 (HDAC3) is a highly conserved, ubiquitously expressed protein that forms multiprotein corepressor complexes to repress gene transcription. Recent studies show that HDAC3 may play a role in cell proliferation. Altered HDAC3 level increases G(2)/M cells, but the mechanism remains unknown. Here we show for the first time, to our knowledge, that the HDAC3 complex, including nuclear receptor corepressor (N-CoR), transducin-beta-like protein 1 (TBL1), and TBL1-related protein 1 (TBLR1), is localized on the mitotic spindle. Knockdown of HDAC3 or N-CoR resulted in a collapsed mitotic spindle that was surrounded by chromosomes arranged in a dome-like configuration. Treatment of mitotic cells with Trichostatin A, an HDAC inhibitor, resulted in similar spindle defects independent of transcriptional regulation. In addition, wild-type HDAC3 but not a deacetylase-dead mutant HDAC3 rescued the phenotypes of HDAC3-depleted cells, suggesting that the enzymatic activity of HDAC3 is important for proper spindle function. Whereas the kinetochores and the spindle assembly checkpoint appeared intact in HDAC3-deficient cells, kinetochore-microtubule attachments were impaired because spindle microtubules were unstable in response to cold treatment. These data suggest that the HDAC3 complex is involved in the formation of functional mitotic spindles and proper kinetochore-microtubule attachment. The level or distribution of acetylated alpha-tubulin was not altered in HDAC3-deficient cells. Taken together, our studies raise the interesting possibility that acetylation-deacetylation of mitotic spindle components may be essential for mitotic spindle function.