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Plumbagin is a plant naphtoquinone exerting anti-cancer properties including apoptotic cell death induction and generation of reactive oxygen species (ROS). The aim of this study was to elucidate parameters explaining the differential leukemia cell sensitivity towards this compound. Among several leukemia cell lines, U937 monocytic leukemia cells appeared more sensitive to plumbagin treatment in terms of cytotoxicity and level of apoptotic cell death compared to more resistant Raji Burkitt lymphoma cells. Moreover, U937 cells exhibited a ten-fold higher ROS production compared to Raji. Neither differential incorporation, nor efflux of plumbagin was detected. Pre-treatment with thiol-containing antioxidants prevented ROS production and subsequent induction of cell death by apoptosis whereas non-thiol-containing antioxidants remained ineffective in both cellular models. We conclude that the anticancer potential of plumbagin is driven by pro-oxidant activities related to the cellular thiolstat.