Vobtusine is an aspidosperma-aspidosperma alkaloid isolated from alkaloid DCM base fraction of the bark of Voacanga foetida (Blume) Rolfe (Apocynaceae). In this study, the effect of vobtusine on the cell cycle, apoptosis induction, and Bcl-2 family protein expression were investigated by flow cytometr, DNA fragmentation analysis, and western blotting, respectively. The results of cell cycle analysis indicated the ratio of the number of cells in each phase did not have significant differences depend on vobtusine concentration although cell number in G1 phase had tendency to decrease according to the increasing of vobtusine concentration. Besides, the sub-G1 phase population of HL-60 cells treated with vobtusine was increased compared with that of cells without treatments (5.9%-23.8%). DNA fragmentation was observed from 20 gM, and the degree of fragmentation was dependent on vobtusine concentration. Caspase-3 activity increased 4.6 times compared to control. After being treated with caspase-9 inhibitor, vobtusine-induced elevation of caspase-3 activity decreased. This shows that caspase-3 activity depends on caspase-9. Vobtusine was also induced apoptotic cell death involved a mitochondrial by Bid activation and Bcl-xL downregulation. Therefore, vobtsusine-induced apoptosis process was initiated by caspase-9 via change of Bcl-2 family protein expression and executed by caspase-3, followed by cell death due to their proteolytic activity. These results indicated the mechanism action of vobtusine as anti-cancer compound via intrinsic pathway. © 2020. All Rights Reserved.