Tylophorine B exhibits 60% inhibition against tobacco mosaic virus (TMV) at a concentration of 1.0 x 10(-6) g/ml. In our study, high affinity for TMV RNA and assembly origin of TMV RNA (oriRNA) was revealed, accompanied by the conformational change of RNA. Considering that TMV assembly begins with the specific recognition by the coat protein aggregate of oriRNA, and that tylophorine B has favorable interaction with oriRNA, we speculate that tylophorine B likely exerts its virus inhibition by binding to oriRNA and interfering with virus assembly initiation. This work may shed light on the possible molecular inhibition mechanism against TMV by tylophorine B, and provide clues in rational design of sequence-specific RNA binding antivirus drugs.