To develop new anticancer agents, we prepared a sulfated polysaccharide (SCGLP1) from the fruiting bodies of Ganoderma lucidum, and the effect of SCGLP1 on human osteosarcoma MG63 cell line was investigated. Our result showed that treatment with SCGLP1 resulted in a significant inhibitory effect on cell proliferation and cell viability of MG63 cells in a dose- and time-dependent manner and caused apoptotic death in MG63 cells through an increase in G0/G1 phase arrest, but had minor cytotoxic effect on human normal osteoblast (NHOst) cells. Western blot analysis identified that SCGLP1-induced apoptosis was associated with an increased protein expression of proapoptotic Bax and Bad, decreased expression of antiapoptotic Bcl-2 and Bcl-XL, loss of mitochondrial membrane potential (Δψm), the release of mitochondrial cytochrome c to cytosol, and cleavage of caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). In addition, pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the SCGLP1-induced apoptosis in MG63 cells. The data indicate that SCGLP1-induced apoptosis is primarily associated with caspase-3- and caspase-9-dependent apoptotic pathway.