Abstract: Objective To study the mechanism of bufalin on inducing cell apoptosis in human lung adenocarcinoma (A549) cells. Methods The growth inhibition was analyzed by MTT assay；The morphological changes in the samples dyed in wright-Giemsa were observed using light microscope.Apoptosis and Mitochondrial membrane potential(ΔΨm) was evaluated by flow cytometry,Western blot analysis was used to evaluate the level of Bcl-2 and Caspase-3 expression. Results (1) Bufalin inhibits cell growth, and 50% inhibitory concentrations(IC₅₀) of the agent at 48h and 72h were (56.14±6.72)nmol/L,(15.57±4.28)nmol/L,respectively.(2) Apoptosis of A549 cells could be efficiently induced by bufalin at different concentrations.(3) A significantly reduction in the Mitochondrial membrane potential(ΔΨm) was induced by bufalin.(4) The expression of Bcl-2 protein was downregulated and Caspase-3 protein was activated during bufalin-induced apoptosis in A549 cells. Conclusion Bufalin-induced lung adenocarcinoma cells apoptosis is through mitochondria signaling pathway in A549 cells.