Genistein is an important chemopreventive agent against atherosclerosis and cancer. However, whether genistein is effective in the treatment of lung cancer, and its underlying mechanism, remains to be determined. The present study demonstrated that genistein treatment of A549 lung cancer cells decreased viability in a dose- and time-dependent manner, and induced apoptosis. Additionally, A549 cells exhibited significantly increased reactive oxygen species formation and cytochrome-c leakage, and activated caspase-3, B-cell lymphoma 2-associated X protein and apoptosis inducing factor expression levels, which are involved in the mitochondrial apoptosis pathway. Furthermore, the phosphatidylinositol-4,5-biphosphate 3-kinase (PI3K)/protein kinase B (AKT)/hypoxia-inducible factor-1 alpha (HIF-1 alpha) and nuclear factor-kappa B (NF-kappa B)/cyclooxygenase-2 (COX-2) signaling pathways were significantly downregulated by genistein treatment. In conclusion, reduced proliferation and increased apoptosis in A549 lung cancer cells was associated with inhibition of the PI3K/AKT/HIF-1 alpha/ and NF-kappa B/COX-2 signaling pathways, which implicates genistein as a potential chemotherapeutic agent for the treatment of lung cancer.