ICP0 is a multifunctional protein that plays diverse roles in herpes simplex virus type 1 (HSV-1) infection. It can promote the lytic replication of HSV-1 and activate a variety of viral or cellular genes when introduced into cells by transfection or infection. However, the exact mechanism of ICP0 action is not fully understood. In the present study, we observed the co-localization of ICP0 and PCAF (P300/CBP-associated factor), a component of histone acetyltransferase (HAT), in the ND10 (nuclear dot 10) nuclear body. We further confirmed the interaction between ICP0 and PCAF via yeast two-hybrid assay, co-immunoprecipitation, and histone acetyltransferase assays. Analysis of the functional significance of this interaction suggested that PCAF improved the ability of ICP0 to activate transcription of viral genes. Using chromatin immunoprecipitation (ChIP) assays, we observed ICP0-enhanced histone acetylation levels in both viral and cellular gene promoters. Our study suggests that ICP0 regulates transcription through specific interaction with PCAF.