Royal jelly acid, 10-hydroxy-trans-2-decenoic acid (10H2DA), is a major lipid component of royal jelly, which is the exclusive diet of queen honeybees. Previously, we showed partial inhibition of lipopolysaccharide (LPS)-induced NF-κB activation by 10H2DA. In this study, the ability of 10H2DA to inhibit LPS-induced nitric oxide (NO) production was investigated. LPS-induced NO production and inducible NO synthase (iNOS) gene transcription were inhibited by 10H2DA. LPS-stimulated interferon (IFN)-β production, IFN regulatory factor-1 induction and IFN-stimulated response element activation, which are required for iNOS induction, were unaffected by 10H2DA. IFN-β-induced NO production, however, was significantly inhibited by 10H2DA. Furthermore, IFN-β-induced nuclear factor (NF)-κB activation and tumour necrosis factor (TNF)-α production were significantly inhibited by 10H2DA, and TNF-α-induced NF-κB activation was also inhibited by 10H2DA. These results and our previous study suggest that 10H2DA inhibits LPS- and IFN-β-induced NO production via inhibition of NF-κB activation induced by LPS or IFN-β.