Gardenin B is naturally occurring flavonoid isolated from Baccharis scandens, that shows potent anti-proliferative activity against human cancer cell lines. Although the exact target and binding sites of gardenin B have not yet been determined, the introduction of additional methyl groups could facilitate penetration through the cell membrane and increase the cytotoxicity in vitro. Cell cycle analysis performed in HL-60 cells showed that inhibition of cell viability by gardenin B was caused by a significant cell cycle arrest at the S and G2-M phases and accompanied by an increase in sub-G1 fraction and phosphatidylserine externalization, indicating apoptotic cell death. This methoxyflavonoid promotes the formation of apoptotic bodies and the internucleosomal degradation of DNA, resulting in the formation and eventual release of oligonucleosomal DNA fragments. Apoptosis induced by gardenin B is associated with activation of both the extrinsic and the intrinsic apoptotic pathways of cell death and occurs through a mechanism that is independent of the generation of reactive oxygen species.