Chios gum mastic (CGM) is produced from Pistiacialentiscus L var chia, which grows only on Chios Island inGreece. CGM is a kind of resin extracted from the stem andleaves, has been used for many centuries in manyMediterranean countries as a dietary supplement and folkmedicine for stomach and duodenal ulcers. CGM is knownto induce cell cycle arrest and apoptosis in some cancer cells.This study was undertaken to investigate the alteration of thecell cycle and induction of apoptosis following CGMtreatment of HL-60 cells. The viability of the HL-60 cells wasassessed using the MTT assay. Hoechst staining and DNAelectrophoresis were employed to detect HL-60 cells undergoingapoptosis. Western blotting, immunocytochemistry,confocal microscopy, FACScan flow cytometry, MMPactivity and proteasome activity analyses were alsoemployed. CGM treatment of HL-60 cells was found to resultin a dose- and time-dependent decrease in cell viability andapoptotic cell death. Tested HL-60 cells showed a variety ofapoptotic manifestations and induced the downregulation ofG1 cell cycle-related proteins. Taken collectively, our presentfindings demonstrate that CGM strongly induces G1 cellcycle arrest via the modulation of cell cycle-related proteins,and also apoptosis via proteasome, mitochondrial andcaspase cascades in HL-60 cells. Hence, we provide evidencethat a natural product, CGM could be considered as a noveltherapeutic for human leukemia.