Camalexin is a phytoalexin that accumulates in various cruciferous plants upon exposure to environmental stress and plant pathogens. It was shown that camalexin has potent antitumor properties, but its underlying mechanisms are still elusive. In the present study, we evaluated the effects of camalexin on human leukemic cells and normal polymorph nuclear cells. CCK-8 assay was used to determine cell viability after camalexin treatment. Apoptosis, intracellular reactive oxygen species (ROS) levels, and loss of mitochondrial membrane potential (MMP) were measured by flow cytometry. The activity of SOD, catalase, and ratio of GSH/GSSG were assayed. ER stress and apoptotic signaling pathway was examined by Western blot. Xenograft mice were used to verify the effect of camalexin in vivo. Our results indicated that camalexin inhibited viability of leukemic but not normal polymorph nuclear cells. Furthermore, camalexin induces apoptosis via the mitochondrial pathway in a caspase-dependent manner. We also observed ER stress is located upstream of apoptosis induced by camalexin. Besides, ROS levels, SOD activity, CAT activity, and GSSG levels were significantly enhanced while the GSH level was decreased after treatment of camalexin. In addition, the generation of ROS is critical for the ER stress and apoptosis induced by camalexin. Finally, administration of camalexin suppresses xenograft tumor graft growth without obvious toxicity. Taken together, this study indicates that camalexin exerts antitumor effects against leukemia cells via the ROS-ER stress-mitochondrial apoptosis pathway.