Background: Ginsenoside Rg3 is a steroidal saponin extracted from Panax ginseng with various anti-tumor effects. While very few studies have focused on exploring the cytotoxic effects of Ginsenoside Rg3 in lung cancer treatment via Notch/hairy and enhancer of split 1 (HES1) pathway. We examined the underlying mechanism of action and the anti-tumor effects of Ginsenoside Rg3 in lung cancer cells and attempted to develop it as a systemic treatment strategy for non-small cell lung cancer (NSCLC). Methods: NCI-H1650, H520 and H1963 cells were used for experiment. MTT assay was used to detect cell proliferation. Annexin V/PI assay was used to detect cell apoptosis. Western blotting was used to detect the level of Notch1, Notch2, HES1, p38 MAPK, Caspase-3 and Caspase-9 expressions. Results: Ginsenoside Rg3 inhibited the proliferation of NCI-H1650, H520 and H1963 cells in a time and dose-dependent manner. Furthermore, Ginsenoside Rg3 induced significant cell apoptosis. The level of Notch1, Notch2 and HES1 expressions decreased but the level of p38 MAPK, Caspase-3 and Caspase-9 expressions increased with Ginsenoside Rg3 treatment. Conclusions: Ginsenoside Rg3 could target the Notch/HES1 pathway to cause apoptosis leading to inhibit the proliferation of lung cancer cells and the results suggested a therapeutic potential of Ginsenoside Rg3 in clinical use for lung cancer treatment.