Background: In chemotherapy for treating lung-cancer, pemetrexed, a multi-targeted antifolate cytotoxic agent, has been used in combination with cisplatin. Recently, autophagy has become an important mechanism of cell death (so called as type II programmed cell death). However, the molecular mechanism of pemetrexed and the role of Autophagy in pemetrexed treated lung cancer cell line remains unclear. The purpose of this study was to determine whether peme-trexed treated lung cancer cell induce autophagy and the inhibition autophagy resulted in apoptosis or necrosis. Methods: To measure out the cell viability aga-inst pemetrexed treatment in H460 cells, we performed MTT assay. Then, to detect the pemetrexed-induced autophagy, we performed Acridine orange assay. Also, to measure the cell viability against autophagy inhibition, we per-formed MTT assay. To confirm apoptosis and necrosis after autophagy inhibition we detected Annexin-V/PI assay by use of FACS.Result: The autophagy had induced in Pemetrexedtreated H460 cells 48H later. Also autophagic vacuoles were observed in pemetrexedtreated H460 cells. Pemetrexedinduced autophagy was inhibited by 3-MA, the autophagy specific inhibitor, which was proven by reduced acidic vesicles. Here was markedly increasing apoptosis When the autohpagy was inhibited by 3-MA.Conclusion: Autophagy induction and markedly increasing apoptosis by autophagy inhibition were identified in pemetrexed treated H460 cells.