Benzyl isothiocyanate (BITC) is detected in abundance in Brassica vegetables, and some previous studies have demonstrated that BITC may potentially function as a chemopreventive agent in humans. This study examined whether BITC inhibits lipopolysaccharide (LPS)-induced inflammatory responses in Raw 264.7 macrophages and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema formation. The treatment of macrophages with various concentrations of BITC resulted in a dose-dependent reduction in the LPS-induced secretion of interleukin (IL)-1beta, TNF-alpha, and IL-6 and their corresponding mRNA levels, as well as in the production of nitric oxide and PGE(2). Consistent with these findings, BITC inhibited the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 proteins and their corresponding mRNAs. BITC inhibited LPS-induced phosphorylation and the degradation of the inhibitor of kappaBalpha, translocation of p65 into the nucleus, and the DNA binding activity and transcriptional activity of NFkappaB. Moreover, the LPS-stimulated phosphorylation of extracellular signal regulated kinase (ERK)1/2 and Akt was suppressed by BITC. BITC also inhibited ear edema formation and the protein expression of iNOS and COX-2 in mouse skin treated with TPA. We demonstrate that BITC is a potent anti-inflammatory agent, and the anti-inflammatory properties of BITC may result from the downregulation of NFkappaB signaling.