H9, a novel herbal extract, demonstrated cytotoxicity in A549 non-small cell lung cancer (NSCLC) cell lines. In this study, we investigated whether H9, and/or co-treatment with an anticancer drug, pemetrexed (PEM), inhibited tumor growth in BALB/c nude mice models bearing A549 NSCLC cells. The mice were separated into groups and administered H9 and PEM for 2 weeks. Protein and mRNA levels were detected using western blotting and reverse transcription polymerase chain reaction, respectively; immunohistochemistry (IHC) was also performed on the tumor tissues. H9 and co-treatment with PEM induced the cleavage of proapoptotic factors, such as caspase-3, caspase-8, caspase-9, and poly(ADP)-ribose polymerase (PARP). Expression levels of cell-death receptors involving Fas/FasL, TNF-related apoptosisinducing ligands (TRAIL), and TRAIL receptors were increased by H9 and co-treatment with PEM. Furthermore, analysis of levels of cell-cycle modulating proteins indicated that tumor cells were arrested in the G1/S phase. In addition, the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt survival signaling pathways were inhibited by H9 and co-treatment with PEM. In conclusion, H9 and co-treatment with PEM inhibited tumor growth in BALB/c nude mice models bearing A549 NSCLC cells. These results indicate that H9 and co-treatment with PEM can be used as an anticancer therapy in NSCLC.