Histone deacetylase (HDAC) inhibitors are emerging as promising cancer therapeutics. HDAC inhibitors have been found to induce cellular activities that are strikingly similar to p53-mediated responses to genotoxic stress. For example, HDAC inhibitors induce cell cycle arrest, apoptosis, and cellular senescence. Because at least 11 HDACs are affected by the current HDAC inhibitors, the HDAC critical for tumor cell survival and proliferation remains unknown. Thus, we sought to characterize the distinct roles of HDACs in the p53 pathway. Through the use of stable MCF7 cell lines which inducibly express short hairpin RNA targeting HDAC2, we found that HDAC2 plays important roles in the p53 pathway. Specifically, we found that knockdown of HDAC2 inhibited cellular proliferation in a dose-dependent manner which was also partly p53-dependent. Furthermore, knockdown of HDAC2 induced cellular senescence. Importantly, we found that knockdown of HDAC2 enhanced p53-dependent trans-repression and trans-activation of a subset of target genes. We found that the enhancement was due to increased p53-DNA binding activity but not alterations in p53 stability or posttranslational modification(s). Thus, for the first time, our data suggest that HDAC inhibitors function through the p53 pathway, at least in part, by activating p53-DNA binding activity. [Cancer Res 2007;67(7):3145–52]