OBJECTIVE To assess the antitumor effects of protosappanin B extracted from Lignum Sappan. METHODS Lignum Sappan was sequentially extracted by boiling water and ethyl acetate. The resulting extract was separated by column chromatography, to yield protosappanin B. The compound was then identified by thin-layer chromatography, high-performance liquid chromatography, elemental analysis, and spectrometry (infrared and ultraviolet). The effects on tumor cell viability and growth of purified protosappanin B were evaluated in vitro by trypan blue exclusion and MTT assays, respectively. And the effects of protosappanin B were assessed in vivo, on H22 mouse liver cancer cell invasion and the survival of tumor-bearing mice. RESULTS Protosappanin B (2 mg/mL) reduced the viability of human bladder cancer T24 cells and mouse bladder cancer BTT cells in a time-dependent manner (P < .05) and significantly inhibited the growth of the human colon cancer cell lines HCT-116 and SW-480. IC50 values of 21.32, 26.73, and 76.53 µg/mL were obtained for SW-480, HCT-116, and BTT cells, respectively, after 48 hours of treatment with protosappanin B. In addition, pretreatment of H22 cells with protosappanin B (final concentration = 6.25 mg/mL) resulted in complete inhibition of tumor formation in KM mice. Furthermore, protosappanin B (200 and 300 mg/kg) significantly increased the survival of BTT tumor-bearing T739 mice, at a rate comparable to that of 1 mg/kg mitomycin. CONCLUSIONS Protosappanin B extracted from Lignum Sappan exerts marked antitumor effects both in vitro and in vivo.