Flavonoids, containing mainly kaempferol rhamnohexoside derivatives, were extracted from Gynostemma pentaphyllum (G. pentaphyllum) and their potential growth inhibition effects against H460 non-small cell lung cancer cells was explored and compared to that on A549 cells. The extracted flavonoids were found to exhibit antiproliferation effects against H460 cells (IC50 = 50.2 μg/mL), although the IC50 of H460 is 2.5-fold that of A549 cells (IC50 = 19.8 μg/mL). Further investigation revealed that H460 cells are more susceptible to kaempferol than A549, whereas A549 cell growth is better inhibited by kaempferol rhamnohexoside derivatives as compared with H460. In addition, flavonoids from G. pentaphyllum induced cell cycle arrest at both S and G2/M phases with concurrent modulated expression of the cellular proteins cyclin A, B, p53 and p21 in A549 cells, but not H460. On the contrary, apoptosis and concomitant alteration in balance of BCL-2 and BAX expression as well as activation of caspase-3 were equally affected between both cells by flavonoid treatment. These observations strongly suggest the growth inhibition discrepancy between H460 and A549 following flavonoid treatment can be attributed to the lack of cell cycle arrest in H460 cells and the differences between H460 and A549 cells may serve as contrasting models for further mechanistic investigations.