AIM: To explore the way and mechanism of cell death induced by Fructus Akebiae in non-small cell lung cancer (NSCLC) cells. METHODS: CCK-8, Hochest33342/PI staining, and colony formation experiments were used to detect cell viability and proliferation. Western blot was used to detect the expression of cell death-related proteins ATG5, HMGB1, GPX4, apoptosis-related proteins, and PI3K-Akt signaling pathway-related proteins. Flow cytometry was used to detect the apoptosis rate. DCFH-DA fluorescent probes were used to detect the levels of total intracellular ROS under a fluorescence microscope. RESULTS: Fructus Akebiae significantly inhibited the cell viability and proliferation of human NSCLC cells (P<0.05). The expression of apoptosis-related proteins BAX, Cleaved caspase3, and Cleaved caspase9 in NSCLC cells was significantly increased (P<0.05), while p-PI3K, p-Akt, and BCL2 were markedly decreased (P<0.05). Fructus Akebiae was found to clearly increase the apoptosis rate and the levels of total intracellular ROS (P<0.05). The antioxidant NAC significantly reversed apoptosis, ROS production and regulation of PI3K-Akt pathway related proteins induced by Fructus Akebiae (P<0.05). CONCLUSION: Fructus Akebiae induces apoptosis via regulating ROS-mediated PI3K-Akt signaling pathway in non-small cell lung cancer cells.