AIM: To investigate the active constituents of Lignum Sappan(Caesalpinia sappan L.) on growth-related signaling and cell mitosis. METHOD: The influence of the ethyl acetate(EtOAc) extract of Lignum Sappan and its constituents on growth-related signaling were evaluated by a luciferase assay in cells stably- transfected with NF-κB, STAT1, or STAT3 responsive luciferase reporter plasmid. The inhibitory effect on the cell cycle was determined by flow cytometric analysis. The anti-tumor activities were assessed in vitro and in vivo. RESULTS: The EtOAc extract of Lignum Sappan had inhibitory activities on growth-related signaling and cell mitosis. Three major active compounds were sappanchalcone, brazilin, and butein. Sappanchalcone blocked cell cycle progression in the G2/M phase, brazilin inhibited TNFα/NF-κB signaling, while butein inhibited IL-6/STAT3 signaling, as well as TNFα/NF-κB signaling. The three compounds all demonstrated cytotoxic activities against human tumor cells in vitro. In a S180 tumor cell-bearing mice model, the anti-tumor efficacy of the EtOAc extract was better than the individual compounds acting alone. CONCLUSION: These results indicate that Lignum Sappan contains multiple active compounds with different antitumor activities, which act synergistically to enhance their anti-tumor effects. The EtOAc extract of Lignum Sappan may be better than individual active constituent as a novel medicine for the treatment of cancer. A: To investigate the active constituents of Lignum Sappan (Caesalpinia sappan L.) on growth-related signaling and cell mitosis. METHOD: The influence of the ethyl acetate (EtOAc) extract of Lignum Sappan and its constituents on growth-related signals were evaluated by a luciferase assay in cells stably-transfected with NF-κB, STAT1, or STAT3 responsive luciferase reporter plasmid. The anti-tumor activities were assessed in vitro and in vivo. RESULTS: The EtOAc extract of Lignum Sappan had inhibitory activities on growth-related signaling and cell mitosis. Three major active compounds were sappanchalcone, brazilin, and butein. Sappanchalcone blocked cell cycle progression in the G2 / M phase, brazilin inhibited TNFα / NF- κB signaling, while butein inhibited IL-6 / STAT3 signaling, as well as TNFα / NF-κB signaling. The three compounds all demonstrated cytotoxic activities against human tumor cells in vi tro. In a S180 tumor cell-bearing mice model, the anti-tumor efficacy of the EtOAc extract was better than the individual compounds acting alone. CONCLUSION: These results indicate that Lignum Sappan contains multiple active compounds with different antitumor activities, which act synergistically The enhancer of anti-tumor effects. The EtOAc extract of Lignum Sappan may be better than individual active constituent as a novel medicine for the treatment of cancer.