To investigate the active constituents against osteosarcoma cells in Marsdeniae Tenacissimae Caulis(MTC) decoction.Methods:The chemical constituents in MTC decoction were investigated by macroporous resin column chromatography(CC)，silica gel CC，preparative high performance liquid chromatography(HPLC)，nuclear magnetic resonance(NMR) spectroscopy，and high-resolution mass spectroscopy(HR-MS).Their anti-osteosarcoma activities were evaluated by the CCK-8 method.Results:Thirty-three compounds were isolated and identified.Among them，compounds 1 to 8 and 24 to 28 were isolated from Marsdeniae Tenacissimae Caulis for the first time.Compounds 6，9，21，22，25，26，and 29 to 31 could significantly inhibit the proliferation of osteosarcoma cells MG63 and 143B.Conclusion:Multiple components of MTC decoction had the inhibitory activities against osteosarcoma cells.This study provides a scientific basis for the application and further exploitation of MTC.