Isoeugenol, which is a naturally occurring o-methoxyphenol in a variety of foods and essential oils, is known to have anti-inflammatory effects, although the mechanism is not clear. In the present study, we investigated the effect of isoeugenol on NF-kappaB signaling leading to inducible nitric oxide synthase (iNOS) expression in RAW 264.7 murine macrophages stimulated with lipopolysaccharide (LPS). Isoeugenol markedly inhibited nitric oxide (NO) production in dose- and time-dependent manners. The decrease in NO production was found to correlate with a decrease in iNOS expression, as determined by Western blot analysis and real-time RT-PCR. To characterize further the inhibitory mechanisms of isoeugenol at the transcriptional level, we examined the DNA-binding and transcriptional activities of NF-kappaB. Isoeugenol inhibited NF-kappaB-dependent transcriptional activity and DNA-binding activity by decreasing the nuclear translocation of p65, which is a component of NF-kappaB. In addition, isoeugenol blocked signaling upstream of NF-kappaB activation, such as degradation of I-kappaBalpha and the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK), in LPS-stimulated RAW 264.7 cells. The isoeugenol analogues eugenol and allylbenzene also inhibited LPS-induced NF-kappaB signaling and iNOS expression, albeit with less potency than isoeugenol. These results suggest that isoeugenol and its analogues inhibit NO production and iNOS expression in LPS-stimulated RAW 264.7 cells, and that these effects are mediated, at least in part, by blocking the phosphorylation of ERK1/2 and p38 kinase, degradation of I-kappaBalpha, and activation of NF-kappaB.