Abstract <p><i>Armillaria mellea</i> is a valuable medicinal and edible fungus widely used in China for its health-promoting properties. It exhibits a broad spectrum of pharmacological activities. Extracts from <i>Armillaria mellea</i> have demonstrated cytotoxic effects against a variety of cancer cell lines. These extracts induce apoptosis and inhibit cell proliferation through multiple mechanisms, including the modulation of critical signaling pathways involved in cell survival and programmed cell death. An additional notable advantage of <i>Armillaria mellea</i> is its non-toxicity, particularly in comparison to conventional chemotherapeutic agents, which frequently lead to adverse side effects. The present study aimed to investigate the anti-lung cancer effects of <i>Armillaria mellea</i> mycelium aqueous (AMMA) extract in the A549 cell line. MTT assay, Hoechst 33342, Fluo-4-AM staining, DCFH-DA staining and ELISA were utilized to evaluate changes in cell viability, nuclear apoptosis, Ca²⁺ concentration, intracellular reactive oxygen species (ROS) concentration, lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and the protein expression levels of Caspase-3/-8/-9. The results demonstrated that AMMA effectively reduced the survival rate of A549 cells, promoted nuclear apoptosis, calcium ion, intracellular ROS level, LDH and MDA content, inhibited high levels of CAT and SOD, and activated the Caspase-3/-8/-9 apoptosis pathway, ultimately mediating the death of cancer cells. This study confirmed that AMMA has an effective anti-lung cancer activity via the Caspase-3/-8/-9 pathway. These data also clarify the potential of AMMA as a natural product based therapeutic agent for the treatment of non-small cell lung cancer.</p>