This study investigates the effects of bufotalin, a bufodienolides compound isolated from toad venom, on sensitizing TNF related apoptosis inducing ligand (TRAIL) induced apoptosis of non small lung cancer cell lines, A549 and PC 9. Cells were treated with bufotalin in the presence and absence of TRAIL. The cells viability was observed using WST 1(4 [3 (4 iodophenyl) 2 (4 nitrophenyl) 2H 5 tetrazolio] 1, 3 benzene disulfonate) assay. The TRAIL induced apoptotic signaling proteins were examined by immunoblotting. The results showed that TRAIL alone showed potent cytotoxicity on PC 9 cells, but weakly induced the death of the A549 cells, indicating that A549 cells are a TRAIL resistance cell line and PC 9 cells are a TRAIL sensitive cell line. Bufotalin significantly enhanced death receptor 4 (DR4) induced cell death in the A549 cell line through the activation of caspase 3 and PARP 1. The combination of bufotalin with TRAIL resulted in the down regulation of Bid, c FLIP, and the up regulation of DR4, RB and p53. In contrast, TRAIL dramatically induced cell death and bufotalin has no effect on TRAIL induced PC 9 cell death. The results indicate that bufotalin sensitizes the A549 cells, but not the PC 9 cells to TRAIL induced apoptosis through DR4 induced apoptotic pathway.