This study demonstrates the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to inhibit LPS-induced expression of iNOS gene and activation of NF-ՊB/Rel in RAW 264.7 cells. Immunohisto-chemical staining of iNOS and Western blot analysis showed magnolol to inhibit iNOS gene expression. Reporter gene assay and electrophoretic mobility shift assay showed that magnolol inhibited NF-ՊB/Rel transcriptional activation and DNA binding, respectively. Since p38 is important in the regulation of iNOS gene expression, we investigated the possibility that magnolol to target p38 for its anti-inflammatory effects. A molecular modeling study proposed a binding position for magnolol that targets the ATP binding site of p38 kinase (3GC7). Direct interaction of magnolol and p38 was further confirmed by pull down assay using magnolol conjugated to Sepharose 4B beads. The specific p38 inhibitor SB203580 abrogated the LPS-induced NF-ՊB/Rel activation, whereas the selective MEK-1 inhibitor PD98059 did not affect the NF-ՊB/Rel. Collectively, the results of the series of experiments indicate that magnolol inhibits iNOS gene expression by blocking NF-ՊB/Rel and p38 kinase signaling.