Salvicine, a novel diterpenoid quinone compound, displays potent antitumor activities in vitro and in vivo, which is under Phase II clinical trials for cancer therapy. Our previous studies have shown that salvicine effectively kills multidrug-resistant (MDR) cells and downregulates mdr-1 and P-glycoprotein (P-gp) levels by activation of transcription factor c-Jun in MDR K562/A02 cells. Recent studies have further demonstrated that salvicine-formed reactive oxygen species (ROS) contribute to its induction of cytotoxicity, DNA double strand breaks and apoptosis. In this study, we showed that salvicine induced equal ROS generation and glutathione depletion in both sensitive K562 and MDR K562/A02 cells. Pre-incubation with thiol antioxidants glutathione or N-acetyl-cysteine (NAC, precursor of intracellular glutathione) almost abolished the cytotoxicity of salvicine, which also could be attenuated by the H(2)O(2)-specific scavenger catalase. Moreover, NAC abrogated salvicine-induced DNA double strand breaks and apoptosis. Notably, both H(2)O(2) and vitamin C potentiated the cytotoxicity and apoptotic induction of salvicine in parental K562 and MDR K562/A02 cells, and catalase could remove such potentiation. Furthermore, pretreatment of K562/A02 cells with NAC eliminated P-gp downregulation, JNK phosphorylation and c-Jun activation induced by salvicine. Our data collectively indicate that salvicine-generated ROS contribute to both cell killing and P-gp downregulation in MDR K562/A02 cells, thus extending our prior related studies. This study also opens the possibility of the combination therapy of salvicine and vitamin C in the future.