In this study, we isolated and purified one homogeneous polysaccharide (TRP) from the fruiting bodies of Trametes robiniophila Murrill, and its average molecular weight was estimated to be 8.7 × 10(4) Da. Monosaccharide composition analysis by gas chromatography (GC) indicated that TRP was composed of glucose, galactose, and arabinose in the molar ratio of 4.2:1.10:1.06. Particularly, we evaluated the anti-cancer efficacy of TRP on human osteosarcoma U-2 OS cells in vitro and associated possible molecular mechanism. Our result provided the first evidence that treatment of U-2 OS cells with TRP resulted in a dose- and time-dependent inhibitory effect on cell proliferation of U-2 OS cells and caused apoptotic death. Moreover, TRP induced the apoptosis of U-2 OS cells via a mitochondria-dependent pathway, as evidenced by an increase in Bax/Bcl-2 ratio, a loss of mitochondrial membrane potential (Δψm), release of cytochrome c from the mitochondria to the cytosol, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase (PARP) in U-2 OS cells. In addition, overexpression of metadherin (MTDH), one carcinogene, was inhibited in U-2 OS cells after exposure to TRP for 24 h. Our findings suggested that TRP inhibited the proliferation of human osteosarcoma cancer cells by promoting apoptosis through the intrinsic mitochondrial pathway, as well as inhibition of MTDH expression.