Lung cancer is almost the most common cause of cancer death in the world. Clinically, the conventional therapy to eradicate the cancer cells is chemotherapy but a better drug remains required. In this study, the effects of three bufadienolides, kalantuboside B, kalantuboside A and bryotoxin C, isolated from Kalanchoe tubiflora (Harvey) were evaluated and characterized in CL1-5 highly metastatic human lung cancer cells. Contrary to the apoptosis-promoting activity in other cancer cells, these three bufadienolides did not induce apoptosis in CL1-5 cancer cells. Instead, they activated an autophagy pathway, as indicated by the increase of autophagosomes formation. The induction of autophagy by these three bufadienolides was demonstrated to link to down-regulation of p-mTOR as well as up-regulation of LC3-II, ATG5, ATG7, Beclin-1. Moreover, among these three compounds, kalantuboside B in which a monosaccharide is attached at the bufadienolide aglycon, exhibited a better autophagy induction. Our findings revealed an alternative mechanism of drug action by bufadienolides in lung cancer cells and provided evidence for the possibility of treating highly metastatic human lung cancer through an autophagy pathway.