Various inhibitors of histone deacetylase (HDAC) activity can sensitize drug resistant cancer cells to chemotherapeutic agents. However, the mechanisms underlying such effects of distinct HDAC inhibitors (HDACi) remain poorly understood. Here we show that both the HDACi trichostatin A and valproic acid induced a sensitization of multidrug-resistant cancer cells to the topoisomerase II inhibitor etoposide/VP16. This effect was associated with increased acetylation of certain lysines on histones H3 and H4, including lysine 16 on histone H4 (H4K16). Overexpression of the histone acetyltransferase hMOF, known to target H4K16, was sufficient to mimic HDACi treatment on sensitization and H4K16 acetylation, and importantly, small-interfering RNA (siRNA)-mediated knockdown of hMOF abolished the HDACi-mediated sensitizing effects as well as the increase in H4K16 acetylation. Conversely, siRNA-mediated knockdown of the H4K16 deacetylase SIRT1 mimicked HDACi treatment whereas overexpression of SIRT1 abolished H4K16 acetylation and significantly reduced the sensitizing effects of HDACi. Interestingly, the effects of hMOF on H4K16 acetylation and sensitization to the topoisomerase II inhibitor could be directly counteracted by exogenous expression of increasing amounts of SIRT1 and vice versa. Our study results suggest that hMOF and SIRT1 activities are critical parameters in HDACi-mediated sensitization of multidrug-resistant cancer cells to topoisomerase II inhibitor and increased H4K16 acetylation.