Objective To investigate the in vitroantivirus effect of total flavonoid from Trollius ledebouriReichb (TFTLR).Methods Madin-Darby canine kidney (MDCK) and Human epithelial type 2 (HEp-2) cell lines were used to test the antivirus effect of TFTLR on nine virus subtypes: four H1N1, one H3N2, and four other subtypes prevalent in North China. Tamiflu, Ribavirin and Lianhua Qingwen were used as active comparators. Comprehensive molecular pathway analyses of TFTLR-H1N1 and TFTLR-H3N2 relationships were also conducted.Results TFTLR inhibited MDCK cell lesions induced by H1N1 subtypes (A/FM1/1/47, A/Puerto Rico/8/1934 H1N1, A1/Tianjin Jinnan/15/2009, and A/Brisbane/59/2007) and by the H3N2 Brisbane/10/2009 strain. TFTLR inhibitory concentration (IC)50values against these viruses were 0.13, 0.07, 0.06, 0.14, and 0.07 mg/ml, respectively; and therapeutic index (TI) values were 8.62, 16.0, 18.67, 8.0, and 16.0, respectively. TFTLR showed no effect on parainfluenza virus type 1, herpes simplex virus type 1, respiratory syncytial virus, and coxsackie group B virus type 4. Pathway analysis revealed possible functional therapeutic mechanisms for TFTLR against H1N1 and H3N2 infections.Conclusion TFTLR may represent a potential therapeutic agent against influenza A subtypes H1N1 and H3N2 that are prevalent in North China, and should be investigated further.