Histone deacetylase 1 (HDAC1) is an epigenetic enzyme that regulates key cellular processes， such as cell proliferation， apoptosis， and cell survival， by deacetylating histone substrates. Aberrant expression of HDAC1 is implicated in multiple diseases， including cancer. As a consequence， HDAC inhibitors have emerged as effective anti-cancer drugs. HDAC inhibitor-induced G/G cell-cycle arrest has?been attributed to epigenetic transcriptional changes mediated by histone acetylation. However， the mechanism of G/M arrest remains poorly understood. Here， we identified mitosis-related protein Eg5 (KIF11) as an HDAC1 substrate using a trapping mutant strategy. HDAC1 colocalized with Eg5 during mitosis and influenced the ATPase activity of Eg5. Importantly， an HDAC1- and HDAC2-selective inhibitor caused mitotic arrest and monopolar spindle formation， consistent with a model in which Eg5 deacetylation by HDAC1 is critical for mitotic progression. These findings revealed a previously unknown mechanism of action of HDAC inhibitors involving Eg5 acetylation， and provide a compelling mechanistic hypothesis for HDAC inhibitor-mediated G/M arrest.