Histone deacetylase inhibitors (HDACIs) are a promising new class of anticancer drugs. However, their mechanism of action has not been fully elucidated. Most studies have investigated the effect of HDACIs on the regulation of gene transcription. HDAC inhibition also leads to genomic instability by a variety of mechanisms. This phenomenon, which has been largely overlooked, may contribute to the cytotoxic effects of these drugs. Indeed, HDACIs sensitize DNA to exogenous genotoxic damage and induce the generation of reactive oxygen species. Moreover, HDACIs target mitosis resulting in chromosome segregation defects. Here, we review the effects of HDACI treatment on DNA damage and repair, and chromosome segregation control.