Clemastanin B, 7S,8R,8'R-(-)-lariciresinol-4,4'-bis-O-β-D-glucopyranoside, is one of the major lignans extracted from Isatis indigotica root (IIR). In this study, the anti-influenza activities of clemastanin B were evaluated in vitro. Clemastanin B was found to inhibit different subtypes of human (H1N1, including swine-origin H1N1; H3N2 and influenza B) and avian influenza viruses (H6N2, H7N3, H9N2) at different magnitudes of activity (IC50 0.087-0.72 mg/ml) while this compound was inactive against respiratory syncytial virus (RSV), adenovirus 3 (ADV3), parainfluenza virus 3 (PIV3), enterovirus 71 (EV71) and human rhinovirus (HRV). An apparent virus titer reduction was detected when MDCK cells were treated with clemastanin B after viral infection, particularly at the early stage, and the ribonucleoprotein (RNP) of the influenza virus was retained in the nucleus after treatment with clemastanin B. These results demonstrated that clemastanin B targets viral endocytosis, uncoating or RNP export from the nucleus. Furthermore, treatment with clemastanin B did not easily result in the emergence of viral drug resistance. The effects of clemastanin B demonstrated in this study may promote the antiviral study of IIR, but additional studies are required to define the anti-influenza mechanism(s).