A variety of stimuli can induce cells to undergo apoptosis, with one of the most reproducible inducers being mild oxidative stress following exposure to anticancer agents. Glutathione (GSH) play an important role in the protection of cells against toxic effects of many eletrophilic drugs and chemicals. Modulation of cellular GSH activity levels provides a potentially useful approach to sensitizing tumor cells to eletrophilic anti-cancer compounds. At previous work, we showed that dehydrocrotonin (DHC), a diterpene lactone isolated from the Amazonian plant Croton cajucara, in free and in ß-cyclodextrin complex (DHC/ßCD) form induced decreasing in the GSH level, mitochondrial membrane depolarisation (Dy_m) and increasing in the mitochondrial swelling indicating altered mitochondrial function. In this study, we performed a detailed study of the role of glutathione and N-acethyl-cysteine (NAC) in the DHC and b-cyclodextrin/DHC cytotoxicity on HL60 cells. The leukaemia cells (HL60) were cultured in suspension at 37°C in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU of penicillin/ml and 100 mg of streptomycin/ml in a humidified atmosphere containing 5% CO₂ in air. To assess viability, the cells were seeded (3 x 10⁵ cells/ml) in 96 wells plates and incubated with different concentrations of free DHC and inclusion complex for 72 h in the presence and absence of antioxidants (GSH 1mM, NAC 1mM, GSH and NAC 0,5mM) and GSH depletory (diethyl-maleate, DEM 0,1mM). Cell viability was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT). It was observed no significant changes in the DHC cytotoxicity using HL60 cells glutathione depleted by pre-treatment with DEM (IC₅₀ was maintained around 200mM).  On the other hand, cytotoxicity of the bCD/DHC complex in the presence of the DEM was increased twice (IC₅₀=100mM), demonstrating that to the DHC and its bCD complex presented distinct mechanisms on induction of GSH depletion. These results suggest that DHC and its bCD complex induce cytotoxicity by oxidative stress mechanism, however with different intensities. Based on the mitochondrial alterations, we conclude that DHC and its cyclodextrin complex triggered apoptosis in HL60 cells through mitochondrial pathway with oxidative stress involvement.