OBJECTIVE: To investigate the apoptosis induced by Pteris semipnnata L 5F (PsL5F) in human anaplastic thyroid carcinoma FRO cells and its molecular mechanism. METHODS: Human anaplastic thyroid carcinoma FRO cells were treated with PsL5F, and the growth inhibition rate was evaluated by MTT assay. The cell apoptosis rate was assessed by Annexin V-FITC fluorescence staining and flow cytometry. Intracellular reactive oxygen species (ROS) levels were analyzed by CM-H2DCFDA fluorescence staining and flow cytometry. Mitochondrial membrane potential (MMP) was measured by DiOD6 (3) fluorescence staining and flow cytometry. The levels of Bax, Cyto C, AIF and cleaved PARP were analyzed by Western blotting. The activity of caspase-3 was assayed by caspase-3 colorimetric assay kit. RESULTS: PsL5F has significant growth inhibitory action on FRO cells in dose and time dependent manners. Under the treatment of 100 mg/L of PsL5F, the percentage of apoptotic cells with phosphatidylserine (PS) externalization was gradually increased in time dependent manner. The rise of ROS level in FRO cells was observed as early as 1h after treated with PsL5F. The elevation of intracellular ROS levels and cell apoptosis could be inhibited by glutathione (GSH), a scavenger of ROS. The MMP in FRO cells was gradually reduced by PsL5F, and the reduction of MMP can be inhibited by GSH. Meanwhile, the levels of Bax in fraction of mitochondrial membrane, Cyto C and AIF in fraction of cytosol were gradually increased. PsL5F can cause the increase of caspase-3 activity and cleavage of PARP, a substrate of caspase-3. CONCLUSION: PsL5F can inhibit growth of human anaplastic thyroid carcinoma FRO cells through inducing apoptosis. The rise of ROS levels in FRO cells plays important role as a secondary messenger in apoptosis induced by PsL5F. Mitochondrium is an important target of PsL5F. Cell apoptosis induced by PsL5F in FRO cells was carried out through translocation of Bax to mitochondrial membrane, reduction of MMP, release of Cyto C and AIF from mitochondria to cytosol, and activation of caspases cascade reaction.