Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G₁ phase of the cell cycle or at the G₂/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer.