Abstract: To isolate anticancer substance from Kraft lignin, ginkgo Kraft lignin(KL) was fractionated with different polar solvents, and the anticancer activity of each fraction was studied by MTT method. The components with the most obvious inhibitory effect were purified by medium-pressure preparation liquid chromatography according to the results of anticancer activity determination. The structure of the compounds was determined by mass spectrometry and ¹³C NMR, and the structure-effect relationship was analyzed. The results of FT-IR spectra showed that ginkgo Kraft lignin had a similar structure to that of ginkgo milled wood lignin(MWL). The total phenol content(TPC) of the fractions decreased with the increase of their relative molecular mass. The results of anticancer activity showed that the ether soluble fraction(LC2) had a strongest inhibitory effect on human-derived lung cancer A549 cells with an IC₅₀ of (366.09±6.39) mg/L. Further purification of LC2 revealed that the isolated compound L7 had the best anticancer activity, with an IC₅₀ value of (89.44±2.13) mg/L against the lung cancer A549 cells. The results of mass spectrometric analysis and ¹³C NMR measurement indicated that compound L7 was a β-5 structure coniferyl aldehyde dimer with a 2-propionaldehyde substituent on the phenolic hydroxyl group. The increase of the aldehyde group on the side chain of the phenylpropane unit of the lignin fraction with small molecule could enhance the anticancer activity, while the increase of the ether bond would weaken the anticancer activity.