Abstract: The antitumor constituents were isolated from Pteris semipinnata L. (PsL), a Chinese traditional herb, and screened using tetrazolium salt (MTT) method. Their cytotoxic effects in vitro on several human tumor cell lines were studied. Compounds 5F, 6F, A and the ethanolic extract of PsL (PSE) were shown to have strong cytotoxicity against five cell lines: human liver adenocarcinoma cell line (HePG-II), human lung adenocarcinom a cell line (SPC-A-1), human gastric adenocarcinoma cell line (MGC-803), human nasopharyngeal carcinoma cells in low differentiation (CNE-2Z) and human liver adenocarcinoma cell line (BEL-7402) in different degrees in a dosedependent manner; compound 6F was the most active one, whose IC₅₀ after 72 h treatment for the above five cell lines were 0.343±0.003, 0.115±0.022, 0.590±0.032, 0.328±0.066 and 0.221±0.058 μg·ml^-1, respectively. Compounds A and 5F were less active; no cytotoxicity of compounds 4F and B were detected on the five cell lines. Analysis of the relationship between structure and activity revealed that the antitumor activity portion in the structure is the α,β-methylene cyclopentanone moiety, and the site and number of the hydroxy groups affect the cytotoxicity of these agents significantly.