Boswellia sacra Flueck essential oil is obtained from frankincense obtained from Boswellia sacra trees. The aim of this study was to investigate the in vitro anti-cancer activity of Boswellia sacra (BS) essential oil in CD133+ and CD133- Colo-320 cells. Essential oil was analyzed by GC-MS. CD133+ cells, cancer stem cells, were obtained from the Colo-320 by the MiniMACS system. Cytotoxicity were evaluated with MTT assay. Anti-cancer activity of BS essential oil was investigated by immunocytochemistry. 41 compounds were identified in the essential oil. 1:2 dilution BS essential oil was found to be active against all the cells for 24 h incubation. Î²-catenin immunoreactivity was significantly decreased in both BS essential oil-treated CD133+ and CD133- Colo-320 cells. Caspase-3 immunoreactivity significantly increased in BS essential oil-treated CD133- Colo-320 cells. We conclude that BS essential oil decreased Î²-catenin signaling molecules which play a crucial role in cancer cell proliferation. BS essential oil promoted apoptosis in CD133- Colo-320 cells.