Tip60 is an essential acetyltransferase required for acetylation of nucleosomal histones and other nonhistone proteins. Tip60 acetylates the p53 tumor suppressor at lysine 120 (K120), a modification essential for p53-dependent induction of PUMA and apoptosis. It is known that Tip60 is turned over in cells by the ubiquitin-proteasome system. However, the deubiquitinase activity for stabilizing Tip60 is unknown. Here we show that USP7 interacts with and deubiquitinates Tip60 both in vitro and in vivo. USP7 deubiquitinase activity is required for the stabilization of Tip60 in order to operate an effective p53-dependent apoptotic pathway in response to genotoxic stress. Inhibiting USP7 with the small-molecule inhibitor P22077 attenuates the p53-dependent apoptotic pathway by destabilizing Tip60. P22077, however, is still cytotoxic, and this is partly due to destabilization of Tip60 © 2013, American Society for Microbiology. All Rights Reserved.