Background and objective Bufalin, a component of the Chinese medicine chan'su, induces apoptosis in various human tumor cell lines. The aim of this study is to investigate the mechanism of anti-proliferation and the induction of apoptosis of Bufalin on non-small cell lung cancer cell. Methods The effect of Bufalin on A549 cell proliferation was measured using MTT assay; the morphological change was observed with Wright-Giemsa stain; cell cycle phase distribution and hypodiploid DNA were determined by flow cytometry with PI; expressions of Livin, Caspase-3 were detected by Western blot. Results MTT assay showed that Bufalin inhibited A549 cell proliferation in time and dose-dependent manner. The inhibition concentration of 50% cell growth (IC50) at 48 h, 72 h and 96 h were (56.14±6.72) nmol/L, (15.57±4.28) nmol/L and (7.39±4.16) nmol/L, respectively; apoptosis of A549 cells could be effectively induced by Bufalin; apoptotic bodies and sub-G1 peak (representing apoptosis) emerged when cells were treated with different concentrations, compared with the untreated control group (P < 0.01). The expression of Livin protein was downregulated (P < 0.01) and Caspase-3 protein was activated during Bufalin-induced apoptosis in A549 cells. Conclusion Bufalin suppresses the growth of A549 cells and induces apoptosis; inhibition of Livin and activation of Caspase-3 may be involved in its mechanism.