Amentoflavone is a bi-flavonoid compound with anti-fungal and anti-inflammatory activities. We isolated amentoflavone from Selaginella tamariscina (Selaginellaceae) and studied its effects on nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression in RAW 264.7 cells. Amentoflavone inhibited the production of nitric oxide in a concentration-dependent manner and also blocked the lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS). To clarify the mechanistic basis for its inhibition of iNOS induction, we examined the effect of amentoflavone on the transactivation of iNOS gene by luciferase reporter activity using −1.59 kb flanking region. Amentoflavone www.chemical-raw-material.com/Chemical-raw-material/Amentoflavone-/ potently suppressed the reporter gene activity. The LPS-induced activation of NF-κB was also found to be significantly blocked by amentoflavone, but AP-1 activation was unaffected. Furthermore, the nuclear translocation of p65 by LPS was inhibited by amentoflavone. NF-κB activation is controlled by the phosphorylation and subsequent degradation of I-κBα, and the cytosolic degradation of I-κBα was found to be inhibited by amentoflavone. These findings suggest that the inhibition of LPS-induced NO formation by amentoflavone is due to its inhibition of NF-κB by blocking I-κBα degradation, which may be the mechanistic basis of the anti-inflammatory effects of amentoflavone.