An ethanol extract from heat-processed Gynostemma pentaphyllum showed more potent cytotoxic activity against human lung adenocarcinoma A549 cells than that of raw G. pentaphyllum. Four constituents were isolated from heat-processed G. pentaphyllum using resin HP-20, silica gel and reversed ODS column chromatography. They were identified by mass and NMR spectra as damulin A and damulin B, gypenoside L and gypenoside LI, respectively. To evaluate the efficacy of these four constituents, the MTT cytotoxicity assay was performed using A549 cells. Based on the structure of these four constituents, the results indicate that the hydroxyl group in C-2 and double bond in C20(21) and C20(22) positions are of importance in inhibition of A549 cell proliferation.