Inhibitors of histone acetyltransferases (HATs) are perceived to treat diseases like cancer, neurodegeneration, and AIDS. On the basis of previous studies, we hypothesized that Cys(1438) in the substrate binding site could be targeted by Δ(12)-prostaglandin J(2) (Δ(12)-PGJ(2)), a cyclopentenone prostaglandin (CyPG) derived from PGD(2). We demonstrate here the ability of CyPGs to inhibit p300 HAT-dependent acetylation of histone H3. A cell-based assay system clearly showed that the α,β-unsaturation in the cyclopentenone ring of Δ(12)-PGJ(2) was crucial for the inhibitory activity, while the 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), which lacks the electrophilic carbon (at carbon 9), was ineffective. Molecular docking studies suggested that Δ(12)-PGJ(2) places the electrophilic carbon in the cyclopentenone ring well within the vicinity of Cys(1438) of p300 to form a covalent Michael adduct. Site-directed mutagenesis of the p300 HAT domain, peptide competition assay involving p300 wild type and mutant peptides, followed by mass spectrometric analysis confirmed the covalent interaction of Δ(12)-PGJ(2) with Cys(1438). Using biotinylated derivatives of Δ(12)-PGJ(2) and 9,10-dihydro-15-deoxy-Δ(12,14)-PGJ(2), we demonstrate the covalent interaction of Δ(12)-PGJ(2) with the p300 HAT domain, but not the latter. In agreement with the in vitro filter binding assay, CyPGs were also found to inhibit H3 histone acetylation in cell-based assays. In addition, Δ(12)-PGJ(2) also inhibited the acetylation of the HIV-1 Tat by recombinant p300 in in vitro assays. This study demonstrates, for the first time, that Δ(12)-PGJ(2) inhibits p300 through Michael addition, where α,β-unsaturated carbonyl function is absolutely required for the inhibitory activity.