The mushroom Inonotus obliquus (Fr.) Pilát (Hymenochaetaceae), has been traditionally used for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes in Russia, Poland and most of Baltic countries. This study was designed to investigate the anti-inflammatory and anti-nociceptive effects of the methanol extract from Inonotus obliquus (MEIO) in vivo and in vitro. MEIO (100 or 200 mg/(kgday), p.o.) reduced acute paw edema induced by carrageenin in rats, and showed analgesic activity, as determined by an acetic acid-induced abdominal constriction test and a hot plate test in mice. To reveal the mechanism of the anti-inflammatory effect of MEIO, we examined its effect on lipopolysaccharide (LPS)-induced responses in a murine macrophage cell line RAW 264.7. MEIO was found to significantly inhibit the productions of nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) in LPS-stimulated RAW 264.7 macrophages. Consistent with these observations, MEIO potently inhibited the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, MEIO inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB), and this was associated with the prevention of inhibitor kappaB degradation and a reduction in nuclear p65 protein levels. Taken together, our data indicate that the anti-inflammatory and anti-nociceptive properties of MEIO may be due to the inhibition of iNOS and COX-2 expression via the down-regulation of NF-kappaB binding activity.