Aim: Investigation of the influence of parthenolide (PTL) on cytarabine (Ara-C) action in an acute promyelocytic leukemia HL-60 cell line and the role of oxidative stress in its action. Methods: Cytotoxic effects and apoptosis were examined using propidium iodide (PI) or PI/Annexin-V staining, respectively. Cells were analyzed using flow cytometry. Results: PTL dose-dependently increased the percentage of dead cells. Cytotoxic and proapototic actions of PTL depend on the level of free radicals, because the addition of N-acetyl-L-cysteine (NAC) to incubation significantly reduced the effect of PTL. We did not observe a similar influence of NAC in the case of Ara-C activity. PTL significantly increased the cytotoxic and proapoptotic effects of Ara-C in comparison to the cytostatic drug alone. Interactions between the investigated compounds were mostly synergistic. NAC significantly reduced the cytotoxic effect of the combination of PTL plus Ara-C. Conclusion: PTL can significantly increase the cytotoxic and proapoptotic effects of Ara-C in an HL-60 cell line and this may warrant further research to improve the efficacy of acute promyelocytic leukemia therapy.