Sulforaphane (SFN), an aliphatic isothiocyanate, is a known cancer chemopreventive agent. Aiming to investigate anti-inflammatory mechanisms of SFN, we here report a potent decrease in lipopolysaccharide (LPS)-induced secretion of pro-inflammatory and pro-carcinogenic signaling factors in cultured Raw 264.7 macrophages after SFN-treatment, i.e. nitric oxide (NO), prostaglandin E₂ (PGE₂) and tumor necrosis factor (TNF-). SFN did not directly interact with NO, nor inhibited inducible NO synthase (iNOS) enzymatic activity. Western blot analyses revealed time- and dose-dependent reduction of LPS-induced iNOS as well as Cox-2 protein expression, which was suppressed at the transcriptional level. To reveal the target of SFN beyond its anti-inflammatory action, we performed electrophoretic mobility shift assay (EMSA) analyses of transcription factor-DNA binding. Consequently, nuclear factor-B (NF-B), a pivotal transcription factor in LPS-stimulated pro-inflammatory response, was identified as the key mediator. SFN selectively reduced DNA-binding of NF-B without interfering with LPS-induced degradation of I-B (inhibitor of NF-B) nor with nuclear translocation of NF-B. As SFN can interact with thiol groups by dithiocarbamate formation, it may impair the redox-sensitive DNA-binding and transactivation of NF-B. SFN could either directly inactivate NF-B subunits by binding to essential Cys-residues or interact with glutathione or other redox regulators like thioredoxin and Ref-1 relevant for NF-B function. Our data provide novel evidence that anti-inflammatory mechanisms contribute to sulforaphane-mediated cancer chemoprevention.