Cirsium japonicum DC, a traditional Chinese medicine, has been widely used as an antihemorrhagic and diuretic agent. The objective of this study was to perform quantitative analysis of flavone by reversed-phase HPLC and examine the anticancer activity of C. japonicum DC in the S180 and H22 mice. Cirsium japonicum DC was separated and purified with several chromatography techniques and two flavone compounds, pectolinarin and 5,7-dihydroxy-6,4'-dimethoxyflavone, were isolated. The content of these two compounds in the methanol, ethanol, and aqueous extractions respectively was determined by HPLC as follows: pectolinarin 1.87%, 1.65%, 1.27%; 5,7-dihydroxy-6,4'-dimethoxyflavone: 0.515%, 0.42%, 0.221%. Furthermore, the effect of the two flavones on the anticancer activity in S180 and H22 mice was studied. Our research shows that these two flavones greatly inhibit cancer cell growth. The rate of inhibiting S180 mice was 55.77% at 50 mg kg( - 1), and the rate of life lengthening was 99.13% at 50 mg kg( - 1) in H22 mice.