Ailanthone (AIL), a monomer derived from ailanthus in Chinese medicine, has been demonstrated to have antitumor effects, albeit the underlying mechanism is unknown. Autophagy and ferroptosis are two modes of cell death that have been championed as potential mechanisms implicated in the antitumor effects of various drugs. The present study demonstrated that AIL effectively suppresses the Lewis cell proliferation in non-small cell lung cancer using MTT and colony formation assays. Autophagy and ferroptosis were verified using western blotting, immunofluorescence and ferroptosis detection. Additionally, the findings revealed that regulating the AMPK/mTOR/p70S6k signaling pathway may be the underlying mechanism for the antitumor effect of AIL. The present study established a theoretical foundation for further research into the utilization of AIL as a novel antitumor approach.