The anti-tumor activity of aliphatic in Huperzia serrata was studied. Guided by antitumor activity,the AB-8 macroporous resin,alumina,silica gel,Sephadex LH 20 and pre-HPLC were used to separate and purify the methanol extract of H. serrata.The structures were identified by modern spectroscopic methods.Nine compounds were isolated and identified,which were classified as pentyl-4-oxoicosa-2(Z),6 (Z)-dienoate (1),2-(2-ethoxy-1-hydroxy-2-oxoethyl)-2-hydroxy-4-methylpentanoic acid (2)，ethyl (9Z,12Z)-octadeca-9,12-dienoate (3),(Z)-nonadec-10-enal (4),oleamide (5),trans-octadec-9-enoic acid (6),bacillamidin A (7),(3Z,6Z,9Z)-3,6,9-docosatriene (8),oplopandiol (9).Compounds 1 and 2 were new compounds. Compounds 3-9 were isolated from H. serrata for the first time. The results of HepG2 cells inhibitory activity showed that compounds 1 and 3 had inhibitory effect on HepG2 with IC₅₀ of 41.12 and 80.54 μmol/L, respectively.