Homoisoflavanone-1 is a natural compound that may be extracted from the Chinese medicinal herb Polygonatum odoratum, which has pronounced antioxidant activities. The present study reports that homoisoflavanone-1 significantly inhibited tumor cell growth and induced apoptosis in A549 non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Homoisoflavanone-1 arrested the cell cycle at the G2/M stage, which was associated with an increase in the accumulation of phosphorylated (p-)p38, p38, p53, and p-cyclin dependent kinase (Cdc)2 proteins, as well as a decrease in Cdc2 expression. In addition, treatment with homoisoflavanone-1 increased the levels of active caspase-3 and decreased Poly ADP-ribose polymerase, which was accompanied by a reduction in the B-cell lymphoma-2/Bak ratio and consequently, apoptosis. Furthermore, homoisoflavanone-1 increased the expression of endoplasmic reticulum (ER) stress-related proteins, including PERK, ATF4 and GADD34 in a dose-dependent manner. In conclusion, homoisoflavanone-1 induced apoptosis in A549 cells by regulating the mitochondria-caspase-dependent and ER stress pathways and resulted in G2/M arrest by activating the p38/p53 signaling pathway. These findings suggest that homoisoflavanone-1 extracted from Polygonatum odoratum may function as a cancer-suppressing agent and has potential as a novel therapeutic method against NSCLC.