Cellular senescence leads to an irreversible block of cellular division capacity both in cell culture and in vivo. The induction of an irreversible cell cycle arrest is very useful for treatment of cancer. Histone deacetylases (HDACs) are considered as therapeutic targets to treat cancer patients. HDAC inhibitors repress cancer growth and are used in various clinical trials. Here, we analyzed whether sodium butyrate (NaBu), an inhibitor of class I and II HDACs, induces cellular senescence in neuroblastoma and prostate cancer (PCa) including an androgen-dependent as well as an androgen-independent human PCa cell line. We found that the HDAC inhibitors NaBu and valproic acid (VPA) induce cellular senescence in tumor cells. Interestingly, also an inhibitor of SIRT1, a class HDAC III, induces cellular senescence. Both neuroblastoma and human prostate cancer cell lines express senescence markers, such as the Senescence Associated-β-galactosidase (SA-β-Gal) and Senescence Associated Heterochromatin Foci (SAHF). Furthermore, NaBu down-regulates the proto-oncogenes c-Myc, Cyclin D1 and E2F1 mRNA levels. The mRNA level of the cell cycle inhibitor p16 remains unchanged whereas that of the tumor suppressor p21 is strongly up-regulated. Interestingly, NaBu treatment robustly increases reactive oxygen species (ROS) levels. These results indicate an epigenetic regulation and an association of HDAC inhibition and ROS production with cellular senescence. The data underline that tumor cells can be driven towards cellular senescence by HDAC inhibitors, which may further arise as a potent possibility for tumor suppression.