AbstractOsteosarcoma is the most common primary bone cancer that affects adolescents with early metastatic potential and drastically reduces their long‐term survival rate if pulmonary metastases are detected at diagnosis. The natural naphthoquinol compound deoxyshikonin exhibits anticancer properties, so we hypothesized that it has an apoptotic effect on osteosarcoma U2OS and HOS cells and studied its mechanisms. After deoxyshikonin treatment, dose‐dependent decreases in cell viability, induction of cell apoptosis and arrest in the sub‐G1 phase of U2OS and HOS cells were observed. The increases in cleaved caspase 3 expression and the decreases in X‐chromosome‐linked IAP (XIAP) and cellular inhibitors of apoptosis 1 (cIAP‐1) expressions after deoxyshikonin treatment in the human apoptosis array were identified in HOS cells, and dose‐dependent expression changes of IAPs and cleaved caspase 3, 8 and 9 were verified by Western blotting in U2OS and HOS cells. Phosphorylation of extracellular signal‐regulated protein kinases (ERK)1/2, c‐Jun N‐terminal kinases (JNK)1/2 and p38 expressions in U2OS and HOS cells was also increased by deoxyshikonin in a dose‐dependent manner. Subsequently, cotreatment with inhibitors of ERK (U0126), JNK (JNK‐IN‐8) and p38 (SB203580) was performed to show that p38 signalling is responsible for deoxyshikonin‐induced apoptosis in U2OS and HOS cells, but not via the ERK and JNK pathways. These discoveries demonstrate that deoxyshikonin may be a possible chemotherapeutic candidate to induce cell arrest and apoptosis by activating extrinsic and intrinsic pathways through p38 for human osteosarcoma.