Antinociceptive effect of glyasperin F isolated from Glycyrrhiza inflata extract (GIE) in ICR mice was studied. Oral administration of GIE (1-100 mg/kg) caused a dose-dependent reduction in acetic acid-induced writhing responses. To identify the active antinociceptive compound from the GIE, sub-fractions were obtained from the EtOAc layer of GIE by using a medium pressure liquid chromatography. From the sub-fractions obtained, the sub-fraction, which, when administered orally (10 mg/kg) showed an antinociceptive effect in both the writhing test and second phase of the formalin test was identified as glyasperin F using NMR and MS analyses. Finally, the antinociceptive effect of glyasperin F in mouse models of pain was confirmed. Orally administered glyasperin F (0.1-10mg/kg) showed a dose-dependent antinociceptive effect in both the writhing test and second phase of the formalin test. Taken together, glyasperin F isolated from the GIE may be used as a leading compound for further studies on pain and as a new drug derived from natural products for pain therapy.