BACKGROUND Lithospermum erythrorhizon has long been used in traditional Asian medicine for the treatment of diseases including skin cancer. In this study, hexane extract from the roots of Lithospermum erythrorhizon (LEH) was chemically characterized and its anticancer activity was tested against the most aggressive form of skin cancer. METHODS The in vitro anticancer studies viz. cell growth, cell cycle and apoptosis, and the expression of tumor regulating proteins were analyzed against B16F10 melanoma cells. In addition, C57BL/6 mice models were used to evaluate the in vivo anticancer potential of LEH. Mice were intraperitoneally injected with LEH at doses of 0.1 and 10mg/kg every 3 days. The tumor inhibition ratio was determined after 21 days of treatment and the histopathological analyses of the tumor tissues were compared. Further, LEH was purified and its active compounds were structurally elucidated and identified by NMR spectra and quantified by HPLC analyses. RESULTS LEH effectively inhibits the growth of melanoma cells with an IC(50) of 2.73μg/ml. Cell cycle analysis revealed that LEH increased the percentage of cells in sub-G1 phase by dose dependent manner. LEH exhibited down regulation of anti-apoptotic Bcl-2 family proteins and up regulation of apoptotic Bax protein expression. Importantly, LEH induced cleavage of poly (ADP-ribose) polymerase (PARP) and activated the caspase cascade (caspase 3) with this cleavage mediating the apoptosis of B16F10 cells. LEH treatment at a dose of 10mg/kg for 21 days in experimental mice implanted with tumors resulted in significant reduction of the tumor growth (43%) and weight (36%). Histopathology analysis of LEH treated tumor tissues showed evidence of increased necrotic cells in a concentration dependent manner. Meanwhile, five naphthoquinone compounds [Shikonin (1); Deoxyshikonin (2); β-Hydroxyisovalerylshikonin (3); Acetylshikonin (4) and Isobutyrylshikonin (5)] were purified from LEH and responsible for its anticancer activity. CONCLUSIONS LEH induced apoptosis in B16F10 cells by activation of caspase 3 and inducing sub-G1 cell cycle arrest. LEH exhibited both in vitro and in vivo anticancer activity. Shikonin derivatives in the LEH are responsible for the anticancer activity.