The transcription factor NF-κB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-κB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of κB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IκBα. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-κB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression.