Abstract: In order to interpret resistance mechanism, controlling effect of surfactin against TMV on tobacco and the expression pattern of defense-related genes were investigated. Liquid culture of Bacillus subtilis OKB105 was precipitated by 6 mol/L HCl. The crude extract of lipopeptides was dissolved by methanol and separated by LH20 chromatographic column. The crude extract detected surfactin only by MALDI-TOF-MS and hemolytic activity analysis. Tobacco was infected with TMV after pretreated by 0.01 g/L surfactin, the result showed that surfactin could control TMV efficiently, the controlling effect reached to 70.9%. The expressions of SA-pathway genes PR1, PR3, PR5, and ET-pathway gene ETR1 were obviously increased by real-time PCR test. The results demonstrated that surfactin could elicite ISR (induced systemic resistance) reaction which depend on SA-pathway and ET-pathway.