Shikonin (SHK), a natural naphthoquinone derived from the Chinese medical herb Lithospermum erythrorhizon, induces both apoptosis and necroptosis in several cancer cell lines. However, the detailed molecular mechanisms involved in the initiation of cell death are still unclear. In the present study, caspase-dependent apoptosis was induced by SHK treatment at 1μM after 6h in U937 cells, with increase in DNA fragmentation, generation of intracellular reactive oxygen species (ROS), fraction of cells with low mitochondrial membrane potential (MMP), and in the expression of BH3 only proteins Noxa and tBid. Interestingly, caspase-independent cell death was also detected with SHK treatment at 10μM, observed as increase in SYTOX® Green staining and release of lactate dehydrogenase (LDH). Necrostatin-1 (Nec-1) completely inhibited the SHK-induced leakage of LDH and SYTOX® Green staining. Cell permeable exogenous glutathione (GSH) completely inhibited 1μM SHK-induced apoptosis and converted 10μM SHK-induced necroptosis to apoptosis. Gene expression profiling revealed that 353 genes were found to be significantly regulated by 1μM and 85 genes by 10μM of SHK treatment, respectively. Among these genes, the transcription factor 3 (ATF3) and DNA-damage-inducible transcript 3 (DDIT3) were highly expressed at 1μM of SHK treatment, while tumor necrosis factor (TNF) expression mainly increased at 10μM treatment. These findings provide novel information for the molecular mechanism of SHK-induced apoptosis and necroptosis.