The cyclopentenone 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-gamma (PPARgamma)-dependent and PPARgamma-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-alpha (ERalpha) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ(2) inhibits both 17beta-estradiol (E(2))-dependent and E(2)-independent ERalpha transcriptional activity by PPARgamma-independent mechanism. In addition, 15d-PGJ(2) directly modifies ERalpha protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ(2) into ERalpha, both in vitro and in vivo. Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys(227) and Cys(240)) within the COOH-terminal zinc finger of ERalpha DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ(2). Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ(2) inhibits DNA binding of ERalpha and subsequent repression of ERalpha target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ(2) can block ERalpha function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERalpha DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERalpha transcriptional activity.