An ethanol extract from heat-processedGynostemma pentaphyllum showed more potent cytotoxicactivity against human lung adenocarcinoma A549 cellsthan that of raw G. pentaphyllum. Four constituents wereisolated from heat-processed G. pentaphyllum using resinHP-20, silica gel and reversed ODS column chromatography. They were identified by mass and NMR spectra asdamulin A and damulin B, gypenoside L and gypenosideLI, respectively. To evaluate the efficacy of these fourconstituents, the MTT cytotoxicity assay was performedusing A549 cells. Based on the structure of these fourconstituents, the results indicate that the hydroxyl group inC-2 and double bond in C20(21) and C20(22) positions areof importance in inhibition of A549 cell proliferation.