Histone deacetylases (HDAC) may have a prominent role in the development of cancer and the response to anticancer therapy. However, the therapeutic relevance and tissue specificity of individual HDAC enzymes remain largely unknown. HDAC inhibitors may function as sensitizing agents to chemotherapies that target DNA through their effects on chromatin structure and plasticity. Here, we report a new role for HDAC2 as a regulator of chromatin compaction status and the mediator of HDAC inhibitor-induced sensitization to chemotherapy. The selective depletion of HDAC2 by small interfering RNA led to reduced expression of heterochromatin maintenance proteins and morphologic changes indicative of chromatin decondensation. Furthermore, depletion of HDAC2 but not HDAC1 or HDAC6 was sufficient to sensitize breast cancer cells to topoisomerase inhibitor-induced apoptosis. The levels of HDAC2 expression appear to correlate with the degree of HDAC inhibitor-induced histone acetylation in a surrogate tissue in patients. These data suggest that HDAC2 may be a relevant pharmacologic and biological target for combination therapy involving drugs that target DNA.