6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) is a bioactive compound extracted from a typical Japanese spice, wasabi (Wasabia japonica (Miq.) Matsumura). In the present study, we found that 6-MITC suppressed the expression of cyclooxygenase-2 (COX-2) induced by lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), but did not suppress that induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), in murine macrophage RAW264. Molecular mechanisms were investigated by targeting the transcriptional factors including activator protein-1 (AP-1), CCAAT/enhancer-binding protein delta (C/EBPdelta), CRE-binding protein (CREB) and nuclear factor kappaB (NF-kappaB), which bind to the core element of COX-2 promoter. LPS induced activation of all of these factors and 6-MITC suppressed LPS-induced activation of AP-1, C/EBPdelta, CREB, but not NF-kappaB. IFN-gamma did not induce any activation of these factors, but 6-MITC suppressed IFN-gamma-induced COX-2 expression, suggesting that the upstream region of the core element is linked for this suppression. Finally, TPA stimulated the activation of CREB and AP-1, but 6-MITC did not block TPA-induced COX-2 expression. These results suggest that LPS, IFN-gamma and TPA regulate COX-2 expression through different mechanisms, and 6-MITC acts as a potent inhibitor of COX-2 expression induced by LPS or IFN-gamma.